Composition from Sphaeranthus indicus and Garcinia mangostana for the control of metabolic syndrome

ABSTRACT

The invention relates pharmaceutical/dietary supplement/food ingredient(s) selected from the extract(s), fraction(s), active compound(s) and phytochemical(s) or mixtures thereof derived from  Sphaeranthus indicus  and their compositions, preferably in combination with at least one component selected from the extract(s), fraction(s), active compound(s) and phytochemical(s) or mixtures thereof derived from  Garcinia mangostana . The ingredients and the composition(s) can be used for the control, prevention and treatment of obesity, metabolic syndrome, diabetes and other metabolic disorders, and also to regulate energy expenditure, prevention of atherosclerotic plaques in coronary artery and abdominal aorta, increase insulin sensitivity, improve glucose tolerance, lower triglyceride levels and balance glucose levels in mammals.

FIELD OF INVENTION

The present invention relates to novel pharmaceutical or dietarysupplement compositions comprising atleast one component selected fromthe extract(s), fraction(s), active compound(s), phytochemical(s) ormixtures thereof derived from Sphaeranthus indicus and at least onecomponent selected from the extract(s), fraction(s), active compound(s),phytochemical(s) or mixtures thereof derived from Garcinia mangostana,optionally containing one or more of pharmaceutically and dieticallyacceptable diluents, vehicles, carriers ad actives or mixtures thereof.

The invention further relates to the use of atleast one componentselected from the extract(s), fraction(s) and active compound(s),phytochemical(s) or mixtures thereof derived from Sphaeranthus indicusor their compositions, preferably in combination with at least onecomponent selected from the extract(s), fraction(s) and activecompound(s), phytochemical(s) or mixtures thereof derived from Garciniamangostana for the control, prevention and treatment of metabolicsyndrome or obesity, and/or one or more disease indications related toor associated with metabolic syndrome and metabolic disorders.

The invention also relates to the amelioration of one or more of thebiomarker proteins or metabolic processes related to metabolic syndrome,obesity and other related or associated disease conditions by theSphaeranthus indicus derived component(s) or their compositions.

BACKGROUND OF THE INVENTION

Sphaeranthus indicus belongs to the family Asteraceae. It is also knownas Gorakhmundi. It is a highly branched, strongly-scented annual herbwith winged stem and toothed wings. Leaves are obovate-oblong, narrowedat the base, dentate and serrate. Flowers are compound heads, globoseovoid. The flowering time spans from November to January in Indianconditions. The medicinally useful parts are root, bark, leaves,flowers, and seeds.

The flowering and fruiting heads of the plant, Sphaeranthus indicuscontains3a-hydroxy-5a,9-dimethyl-3-methylene-3a,4,5,5a,6,7,8,9b-octahydro-3H-naphtho[1,2-b]furan-2-one(7-α-Hydroxy-4,11(13)-eudesmadien-12,6-olide or 7-hydroxyfrullanolide)as a major compound. It strongly inhibits pro-inflammatory cytokines.

Several other compounds have been reported from Sphaeranthus indicuslike methyl chavicol, α-ionone, δ-cadinene, p-methoxycinnamaldehyde asmajor constituents and α-terpinene, citral geraniol, geranyl acetate,β-ionone, sphaerene, indicusene and sphaeranthol as minor constituentsof essential oil (Perfum. Essent. Oil Record. 1959, 50, 765; Chem.Abstr. 1960, 54, 798Og); 7α-hydroxyeudesm-4en-6,12-olide, its β-isomer,dihydrolactone, a new sesquiterpene acid, 2-hydroxycostic acid,β-eudesmol and illicic acid (Jayant S. Sohoni et al, J. Chem. Soc.,Perkin Trans. 1, 1988, 157-160); Eudesmanoids like11-alpha-13-dihydro-3alpha,7alpha-dihydroxy-4,5-epoxy-6beta,7-eudesmanolide,11alpha,13-dihydro-7alpha-acetoxy-3beta-hydroxy-6beta,7-eudesm-4-enolideand 3-keto-beta-eudesmol (Pujar P P et al, Fitoterapia. 2000 June;71(3):264-8) and a sesquiterpene glycoside (Shekhani M S et al, 1990;Phytochemistry 29, 2573-2576).

Some of the non-patent literature of Sphaeranthus indicus is quotedbelow:

In a study evaluating the anti-inflammatory effects of Rubia cordifolia,Curcuma longa, Hemidesmus indicus, Aradirachta indica and Sphaeranthusindicus, Sphaeranthus was found to be more potent in suppressing theproinflammatory cytokines interleukin-8 (IL-8) and tumor necrosis factorα (TNF α) induced by the culture supernatant of Propionibacterium acnesin polymorphonuclear leukocytes (PMNL) and monocytes [Jain A et. al.;Phytomedicine. 2003 January; 10(1):34-8].

The petroleum ether extract from the flower heads of Sphaeranthusindicus Linn was found to be effective in increasing phagocyticactivity, hemagglutination antibody titer and delayed typehypersensitivity when tested in mice. The petroleum extract showed adose-response relationship. It was found that 200 mg/kg dose was theoptimum dose. Sphaeranthus acts as an immunomodulatory agent, bystimulating both humoral and cellular immunity as well as phagocyticfunction. [Bafna A R et. al; J Herb Pharmacother. 2007; 7(1):25-37].

In a study, the effect of aqueous extract of Sphaeranthus indicus (300mg/kg/day, i.p) against dexamethasone (10 mg/kg/day, s.c) inducedchanges in lipid profile in rat was investigated. S. indicus showedsignificant decrease in serum total cholesterol, triglyceride, LDL, VLDLand there was no significant change in the level of HDL. Atherogenicindex also reduced significantly after S. indicus treatment thusindicating that, S. indicus has a potential lipid lowering effect [TenpeC R et al; Biomed. Vol. 02 (4), 2008; 400-403].

Recently, in another study, the antihyperglycaemic effects ofSphaeranthus indicus in rats rendered diabetic by nicotinamide [120 mgkg(−1) i.p.] and streptozotocin (STZ) [60 mgkg(−1) i.p] wasinvestigated. Oral administration of S. indicus for 15 days resulted insignificant decrease in blood glucose levels and increases in hepaticglycogen and plasma insulin levels. Fasting normal rats treated with thealcoholic extract of S. indicus showed significant improvement in oralglucose tolerance test. Glibenclamide was used as a reference standard[Prabhu K S et al; J Pharm Pharmacol. 2008; 60(7): 909-16].

None of the above literature describes the amelioration of metabolicsyndrome related biomarkers or its therapeutic effects against metabolicsyndrome or disease conditions associated with metabolic syndrome bySphaeranthus indicus.

Aqueous extract of Sphaeranthus indicus has been used in Tenpe C R et alstudy, which do not contain significant quantities of7-hydroxyfrullanolide, whereas the present inventive compositionscontain lipophilic extract comprising 7-hydroxyfrullanolide as activecompound. Hence the inventive compositions are different from those usedin the Tenpe C R et al study. Similarly, alcoholic extract of roots andstolons has been used in Prabhu K S et al's study. However, the presentinventive compositions are derived from flower heads, which containpredominantly 7-hydroxyfrullanolide and hence are different from thoseused in the Prabhu K S et al's study.

Some of the Patent literature of Sphaeranthus is quoted below:

PCT Publication WO07036900A2 relates to a novel herbal compositioncomprising an extract of flowering and fruiting heads of the plant,Sphaeranthus indicus containing3a-hydroxy-5a,9-dimethyl-3-methylene-3a,4,5,5a,6,7,8,9b-octahydro-3H-naphtho[1,2-b]furan-2-one(7-Hydroxy-4,11(13)-eudesmadien-12,6-olide), as a bioactive marker. Thesaid invention also relates to methods of manufacture of the saidcompositions.

Japanese Patent Publication JP07138180A2 relates to inhibitor ofhyaluronidases containing an extract separated from at least one herbselected from the group consisting of Azadirachta indica, Cymbopogonnardus, Murraya koenigii, Sphaeranthus indicus, Ocimum sanctum,Tinospora cordifolia and Phyllanthus nuriri and its use as a cosmeticcapable of preventing aging and preventing fine wrinkles and dryness ofthe skin.

PCT Publication WO06134609A2 discloses herbal anticancer agentcomprising the extract of plant Sphaeranthus indicus or group ofcompounds obtained from the plant Sphaeranthus indicus. It alsodiscloses a pharmaceutical composition comprising the said agent,methods for preparing the composition, methods of treating all kinds ofcancer in mammals including human beings, methods of making the plantextract and methods for obtaining the active constituents.

PCT Publication WO06016228A2 relates to a compound or group of compoundspresent in an active principle derived from plants of the speciesSphaeranthus, for the preparation of pharmaceutical formulations or foodsupplements for the prophylaxis and/or treatment of tumor diseases. Thesaid invention furthermore relates to a novel method for the isolationof an active principle from Sphaeranthus plant parts which are effectivein prophylaxis and/or treatment of cancers.

U.S. Pat. No. 7,344,738 provides pharmaceutical or medicinal preparationcomprising a combination of two herbal compositions, including onecomprising a mixture of the following herbs: Moringa oleifera, Boerhaviadiffusa, Onosma bracteatum, Bauhinia variegata, Spheranthus indicus,Tecomella undulata, Chlorophytum borivilianum, Ficus racemosa, andCyperus rotundus, or a mixture of the active ingredients that have beenextracted from those herbs or chemically synthesized. The herbalpreparation is effective for the treatment of a wide range ofphysiological and pathological conditions in the human body resultingfrom a weakened or deteriorating immune system.

Garcinia mangostana which is also used in the present novel compositionbelongs to the family Guttiferae. Garcinia consists of roughly 300species of dioecious trees and shrubs distributed into South America(where they are also known as Rheedia), Africa, Madagascar, andSoutheast Asia. Most of the species diversity in the genus is centeredin Malaysia, with over two-thirds of the species in the genus beingfound there [http://www.mobot.org/MOBOT/Research/mangosteen/].

Mangostins are a major class of compounds in Garcinia mangostana. Thestructure of mangostin was established by Peter Yates et al. [PeterYates, George H. Stout; J. Am. Chem. Soc.; 1958; 80(7); 1691-1700].

Several pharmacological activities have been reported for mangosteenlike anti-inflammatory [Gopalakrishnan C et al., Indian J Exp Biol. 1980August; 18(8):843-6], histaminergic and a serotonergic receptor blockingagent [Chairungsrilerd N et al., Planta Med. 1996 October; 62(5):471-2],anti-cancer agent [Ee G C et al., J Asian Nat Prod Res. 2008 May;10(5):481-5], Anti-microbial [Sundaram B. M., et al.; Planta Med. 1983;48:59-60], etc. and a lot of research is being conducted for exploringnew activities.

Some of the non-patent literature of Garcinia is described below:

In an assay guided fractionation study, the different fractionationsfrom lipophilic to hydrophilic using combined solvent extraction andAmberlite XAD2 adsorption chromatography was studied. The mangosteenpericarps extracts have been tested for alpha-amylase inhibitionactivity and it was concluded that xanthones does not have anyinhibiting activity but the Oligomeric Proantho Cyanidins from thehydrophilic fraction are reported to be 56 times more effective ininhibiting alpha-amylase. [Eng Kiat Loo A, Huang D.; J Agric Food Chem.2007 Nov. 28; 55(24):9805-10].

It is of importance to know that in the above stated study [Eng Kiat Looet al, 2007], the alpha-amylase inhibitory activity was attributed tooligomeric proantho cyanidins. It was further stated that xanthonefraction did not have any alpha-amylase inhibition activity. Hence theactive compound(s) responsible for the activity of the extracts orfractions of this prior art article are different from those of thepresent invention.

In another study the investigators determined Aldose Reductase (ALR2)inhibitory effect of G. mangostana. α-Mangostin was found to be potentagainst ALR2. It was concluded that α-mangostin might be useful inpreventing diabetic complications [Sri Fatmawatia et al., Biology,Chemistry, Pharmacology and Clinical Studies of Asian Plants Apr. 9-11,2007, Surabaya, Indonesia].

A cursory review of prior art reveals that, there is no knowledgerelating to the use of the composition comprising Sphaeranthus indicusand Garcinia mangostana for treating Metabolic Syndrome or forameliorating metabolic marker proteins.

Metabolic Syndrome also known as Syndrome X, insulin resistance syndromeand DysMetabolic Syndrome is a condition, wherein a group of diseasedstates, which increase atherosclerosis, stroke and diabetes.

Metabolic Syndrome was first described by Reaven in 1988 [Reaven, (1988)Diabetes 37; 1595-1607] as a cluster of interrelated common clinicaldisorders, including obesity, insulin resistance, glucose intolerance,hypertension and dyslipidemia.

A criteria for diagnosing Metabolic Syndrome was established by TheAdult Treatment Panel-III (ATP-III) of the National CholesterolEducation Program in 2001 [JAMA (2001),285; 2486-2497]. Five Criteriawere selected by this Panel to identify individuals with MetabolicSyndrome including abdominal obesity, impaired fasting glucose, hightriglyceride (TG), low HDL cholesterol (HDL-C) concentrations andincreased blood pressure. Metabolic Syndrome is diagnosed, if any threeof the components are present in an individual.

A lot of research is being carried out over a decade to develop agentsto control Metabolic Syndrome. The application of metabolic markers forthe control of this syndrome has also been attempted.

People with Metabolic Syndrome are at high risk of coronary heartdisease, other diseases related to plaque buildups in artery walls(e.g., stroke and peripheral vascular disease) and type-2 diabetes.

Some of the biological conditions are also considered as markers forMetabolic Syndrome, which include Hyperuricemia—[Vuorinen-Markkola H etal; J Clin Endocrinol Metab. 1994; 78(1):25-9.]; Hypertriglyceridemia[Grundy S M.; Am J Cardiol. 1998; 81(4A):18B-25B]; Hypoadiponectinemia[Stern N et al; J Cardiometab Syndr. 2007; 2(4): 288-94];Microalbuminuria [Brahimi M et al; Arch Mal Coeur Vairs. 2007;100(8):673-6].

Some of the patents are quoted below which refer to the treatment andcuring of Metabolic Syndrome.

PCT Publication WO08086403A1 describes the identification and isolationof chromones and novel chromone compositions from plant sources that areeffective in enhancing adiponectin production by adipocytes andregulating genes involved in fatty acid biosynthesis. The invention alsoinclude methods for the prevention and treatment of a variety ofdiseases and conditions including, but not limited to insulinresistance, glucose intolerance, hyperglycemia, Metabolic Syndromes,dyslipidemia, and hypertriglyceridemia.

PCT Publication WO08074935A2 relates to compositions and products thatcan be obtained from plants, such as extracts, fractions and/ormolecules useful for preventing or treating metabolic disorders, obesityand/or diseases associated therewith such as the X syndrome (MetabolicSyndrome), type-2 diabetes, or for producing food additives for humanbeings or animals.

PCT Publication WO08093848A1 discloses a pharmaceutical productcontaining phosphatidylcholine derived from soybean for oraladministration or for oral cavity application, a functional food and anoral composition which can prevent or ameliorate a disorder in thephysical function induced by the increase in an inflammation marker,which can reduce the occurrence of Metabolic Syndrome or the risk of adisease and Metabolic Syndrome, and which can maintain or promote thehealthy state.

Based on the information cited above and several other documents, theinventors of the present invention have felt the need of an effectivenatural composition which can efficiently be used for the control ofMetabolic Syndrome and several other associated and related diseases.

According to our knowledge, there is no prior art relating to the usageof ingredients selected from the extracts, fractions or activecompounds, phytochemical(s) or mixtures thereof derived fromSphaeranthus indicus and their compositions in combination withingredients selected from the extracts, fractions or active compounds,phytochemical(s) or mixtures thereof derived from Garcinia mangostanafor the amelioration of for the control, prevention and treatment ofdisease conditions associated with or related to obesity, metabolicSyndrome and other metabolic disorders.

SUMMARY OF THE INVENTION

In the primary aspect, the invention provides novelpharmaceutical/dietary supplement/food ingredient compositionscomprising atleast one component selected from the extract(s),fraction(s), active compound(s) and phytochemical(s) or mixtures thereofderived from Sphaeranthus indicus and at least one component selectedfrom the extract(s), fraction(s), active compound(s) andphytochemical(s) or mixtures thereof derived from Garcinia mangostana,optionally containing one or more of pharmaceutically and dieteticallyacceptable phytochemical actives, diluents, vehicles, carriers andactives or mixtures thereof.

In the other primary aspect, the invention provides the use of atleastone pharmaceutical/dietary supplement/food ingredient component selectedfrom the extract(s), fraction(s), active compound(s) andphytochemical(s) or mixtures thereof derived from Sphaeranthus indicusor their compositions, preferably in combination with at least onecomponent selected from the extract(s), fraction(s) and activecompound(s) and phytochemical(s) or mixtures thereof derived fromGarcinia mangostana for the control, prevention and treatment ofmetabolic syndrome or obesity, and/or one or more disease indicationsrelated to or associated with metabolic syndrome.

In the other aspect, the invention provides Sphaeranthus indicus derivedcomponent(s) selected from the extract(s), fraction(s), activecompound(s) and phytochemica(s) or mixtures thereof or theircompositions, preferably in combination with at least one componentselected from the extract(s), fraction(s), active compound(s) andphytochemical(s) or mixtures thereof derived from Garcinia mangostanafor the amelioration of the expression or production of one or morebiological marker proteins related to or associated with metabolicsyndrome, obesity and other disease conditions associated with metabolicsyndrome including but not limited to Matrix Metalloproteinase-1(MMP-1), Matrix Metalloproteinase-3 (MMP-3), Peroxisomeproliferator-activated receptor gamma (PPARγ), Adipose DifferentiationRelated Protein (ADRP), adipocyte CD36, Macrophage CD36, MonocyteChemotactic protein (MCP-1), Oxidized LDL (Ox-LDL), adipocytefatty-acid-binding protein (aP2/FABP4/A-FABP), beta-3 AdrenergicReceptor (β3AR), Perilipin, Adiponectin and Protein tyrosinephosphatase-1B (PTP-1B).

In a further aspect, the invention provides Sphaeranthus indicus derivedcomponent(s) selected from the extract(s), fraction(s), activecompound(s) and phytochemical(s) or mixtures thereof or theircompositions, preferably in combination with at least one componentselected from the extract(s), fraction(s) and active compound(s) derivedfrom Garcinia mangostana for the amelioration of the metabolic processessuch as promotion of lipolysis, and inhibition of adipogenesis,alpha-amylase enzyme and alpha-glucosidase enzyme activities.

In a major important aspect of the invention, pharmaceutical/dietarysupplement/food ingredient(s) the extracts, fraction(s), pure compoundsor phytochemicals or mixtures thereof derived from Sphaeranthus indicusor the compositions comprising the said ingredients for the control,prevention and treatment of metabolic syndrome or obesity, and/or one ormore disease indications related to or associated with metabolicsyndrome.

In other major aspect of the invention the extracts or enrichedfractions or pure compounds or the mixtures thereof derived fromSphaeranthus indicus can be used alone or in combination with one ormore of pharmaceutically or dietically acceptable vehicle or carrier ordiluents or mixtures thereof for the prevention, control and treatmentof metabolic syndrome or obesity and/or one or more disease conditionsrelated to or associated thereof.

In a further aspect of the invention the active compound responsible foractivity of Sphaeranthus indicus for prevention, control and treatmentof metabolic syndrome and obesity and other related and associatedconditions including but not limited to, 7-hydroxyfrullanolide;11α,13-dihydro-3α,7α-dihydroxy-4,5-epoxy-6β,7-eudesmanolide;11α,13-dihydro-7α-acetoxy-3β-hydroxy-6β,7-eudesm-4-enolide;3-keto-β-eudesmol; 11α,13-dihydro-3α,7α-dihydroxyuedes-4-en-6α,12-olide;11α,13-dihydro-3α,7α-dihydroxyfrullanolide;11α,13-dihydro-7α,13-dihydroxyfrullanolide;11α,13-dihydro-7α,-hydroxy-13-methaoxyfrullanolide;2α,7α-dihydroxy-4-en-11,13-dihydroeudesn-6,12-olide; 2α-hydroxycosticacid; 3keto,7α-hydroxyeudesm-4-en-6,12-olide (cryptomeridiol);4-epicryptomeridiol; Sphaeranthanolide; 2α-hydroxysphaerantholide;2α-Acetoxysphaerantholide; 2α,7α-Dihydroxysphaerantholide;2α-Acetoxy-7α-hydroxysphaerantholide;2α-Acetoxy-5α-hydroxyisosphaerantholide etc., preferably7-hydroxyfrullanolide or related compounds or its analogs or mixturesthereof.

In the other aspect, the source of 7-α-hydroxy-4,11(13)-eudesmadien-12,6-olide (7-hydroxyfrullanolide) used in thepresent invention can be Sphaeranthus indicus or any plant source orsynthesis.

In other aspect of the invention, the Sphaeranthus indicus derivedcomponents and their compositions can be used effectively for theprevention, treatment and control of one or more conditions selectedfrom but not limited to Metabolic Syndrome, obesity, atherosclerosis,diabetes, insulin resistance, regulate energy expenditure, prevention ofatherosclerotic plaques in coronary artery and abdominal aorta, increaseinsulin sensitivity, improve glucose tolerance, lower triglyceridelevels and balance glucose levels in mammals.

In another aspect, the present invention provides compositionscomprising at least one component selected from the extract(s),fraction(s), active compound(s) and phytochemical(s) or mixtures thereofderived from Sphaeranthus indicus in combination with at least onecomponent selected from the extract(s), fraction(s), active compound(s)and phytochemical(s) or mixtures thereof derived from Garciniamangostana to produce synergistic effects to prevent or control or treatmetabolic syndrome or disease conditions associated with metabolicsyndrome, and for amelioration of the production of different biologicalmarker proteins associated with metabolic syndrome or disease conditionsassociated with metabolic syndrome.

In the other aspect, the invention provides methods for the prevention,treatment and control of Metabolic Syndrome, obesity and other diseaseconditions associated with or related to metabolic syndrome, especially,diseases and conditions mediated by insulin resistance in mammals,wherein the method comprises of administering to a subject in needthereof an effective amount of a pharmaceutical or dietary supplementselected from the extracts, fractions, active compounds andphytochemicals(s) or mixtures thereof derived from Sphaeranthus indicusoptionally containing atleast one component selected frompharmaceutically or dietetically acceptable vehicles, carrier, diluentand actives and mixtures thereof.

In the other aspect, the invention provides methods for the prevention,treatment and control of Metabolic Syndrome, obesity and other diseaseconditions associated with or related to metabolic syndrome, especially,diseases and conditions mediated by insulin resistance in mammals,wherein the method comprises of administering to a subject in needthereof an effective amount of a pharmaceutical or dietary supplementselected from the extracts, fractions, active compounds andphytochemical(s) or mixtures thereof derived from Sphaeranthus indicus,preferably in combination with atleast one component selected from theextract(s), enriched fraction(s) or pure compound(s) derived fromGarcinia mangostana, optionally containing atleast one componentselected from pharmaceutically or dietetically acceptable vehicles,carrier, diluent and actives and mixtures thereof.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Illustrates Bar diagram which shows percent reduction in MMP-1in A2058 human melanoma cell culture supernatants obtained bySphaeranthus indicus ethyl acetate extract (LI/DD-II/054A/01). A2058cells were induced with 50 nM PMA in absence or presence of differentconcentrations of LI/DD-II/054A/01 for 24 h as indicated. Secreted MMP-1concentration in the cell free culture supernatants was measured usingMMP-1 ELISA Development Kit (R&D System, Minneapolis, Minn., USA). TheMMP-1 concentration in culture supernatants was estimated quantitativelyfrom the standard curve generated using known concentrations of MMP-1.Percentage of MMP-1 inhibition at each concentration of test compoundwas calculated from the formula: {(Conc. of MMP-1 in PMA induced−Conc.of MMP-1 in the test well)×100}+Conc. of MMP-1 in PMA induced wells.

FIG. 2: Illustrates Bar diagram which shows percent reduction in MMP-3concentration in A549 human lung tumor cell culture supernatantsobtained by Sphaeranthus indicus ethyl acetate extract(LI/DD-II/054A/01). A549 cells were induced with 10 ng/ml human IL-1β inabsence or presence of different concentrations of LI/DD-II/054A/01 for24 h as indicated. Secreted MMP-3 concentration in the cell free culturesupernatants was measured using MMP-3 ELISA Development Kit (R&D System,Minneapolis, Minn., USA). The MMP-3 concentration in culturesupernatants was estimated quantitatively from the standard curvegenerated using known concentrations of MMP-3. Percentage of MMP-3inhibition at each concentration of test compound was calculated fromthe formula: {(Conc. of MMP-3 in IL-1β induced−Conc. of MMP-3 in thetest well)×100}÷Conc. of MMP-3 in IL-1β induced wells.

FIG. 3: Illustrates the modulating of marker proteins of Adipogenesisand lipolysis processes in 3T3-L1 adipocytes by Sphaeranthus indicusethyl acetate extract (LI/DD-II/054A/01) and 7-hydroxyfrullanolide(LI054A01). Representative immuno blots indicate down-regulation ofvarious marker proteins such as PPARγ (A), ADRP (B), CD36 (C), aP2 (D),β3AR (E) and perilipin (F). The 3T3-L1 mouse pre-adipocytes were allowedto differentiate in absence or presence of various concentrations ofLI/DD-II/054A/01 or LI054A01 as indicated. Vehicle control culturesreceived similar concentrations of DMSO only. Expression of actinprotein was evaluated in each blot as the internal control. Expressionof each protein was measured densitometrically and normalized with actinexpression. The comparative levels are represented as bar diagrams (sidepanels).

FIG. 4: Illustrates down-regulation of high glucose induced CD36expression in macrophage cells by Sphaeranthus indicus ethyl acetateextract (LI/DD-II/054A/01) and 7-hydroxyfrullanolide (LI054A01). TheJ774 mouse macrophage cells were exposed to high glucose (600 mg/dL) for5 days in presence or absence of LI/DD-II/054A/01 at variousconcentrations or 1 μg/ml of LI054A01 as indicated. The control culturesreceived low glucose (100 mg/dL). Representative immuno blot assaydemonstrates down regulation of CD36 protein. The expression of actinprotein is considered as the internal control. Bar diagram shows theCD36 expression normalized with actin protein (lower panel).

FIG. 5: Representative immuno blots showing the modulations of PPARγ(A), ADRP (B), aP2 (C), CD36 (D), perilipin (E) and β3AR (F) proteinexpressions in 3T3-L1 adipocytes treated with the methanol extract ofGarcinia mangostana (AR 933) as indicated. Protein expressions weredensitometrically analyzed and normalized with the actin expression. Bardiagram in each panel shows normalized protein expressions in arbitraryunits. In bar diagrams, the bars represent protein expression in cellstreated with vehicle control (a), 2.5 μg/ml (b) and 5.0 μg/ml (c) of AR933, respectively.

FIG. 6: Representative immuno blots show the modulations of PPARγ (A),ADRP (B), aP2 (C), CD36 (D), perilipin (E) and (β3AR (F) proteinexpressions in 3T3-L1 adipocytes treated with either Sphaeranthusindicus ethyl acetate extract (LI/DD-II/054A/01) or Garcinia mangostanamethanol extract (AR 933) or composition 1B comprising these twoextracts as indicated. Protein expressions were densitometricallyanalyzed and normalized with the actin expression. Bar diagram in eachpanel shows normalized protein expressions in arbitrary units. In bardiagrams, the bars represent protein expressions in cells treated withvehicle control (a), LI/DD-II/054A/01 (b), AR 933 (c) and composition 1B(d).

FIG. 7: Representative immunoblots showing down-regulation ofatherosclerotic markers in high glucose induced macrophage cells treatedwith either Sphaeranthus indicus ethyl acetate extract(LI/DD-II/054A/01) or Garcinia mangostana methanol extract (AR 933) orcomposition 1B. Immuno blots show down regulation of CD36 (A), MCP-1 (B)and Ox-LDL (C) protein expression. Expression of actin protein isconsidered as the loading control. Bar diagrams in respective panelsshow the normalized protein expression. Bars represent expression levelsfor a, Glucose 100 mg/dL; b, Glucose 600 mg/dL; c, LI/DD-II/054A/01 (5μg/ml); d, AR 933 (5 μg/ml), and e, composition 1B (5 μg/ml).

FIG. 8: Representative immunoblot showing over expression of adiponectinprotein in 3T3-L1 adipocytes treated with 5 μg/ml of eitherLI/DD-II/054A/01 or AR 933 or composition 1B treated. Proteinexpressions were densitometrically analyzed and normalized with theactin expression. Bar diagram in each panel shows normalized proteinexpressions in arbitrary units. In bar diagrams, the bars representprotein expressions in cells treated with vehicle control (a),LI/DD-II/054A/01 (b), AR 933 (c) and composition 1B (d).

FIG. 9: Representative immunoblot showing down regulation of PTP-1Bprotein expression in 3T3-L1 adipocytes treated with 5 μg/ml of eitherLI/DD-II/054A/01 or AR 933 or composition 1B. Protein expressions weredensitometrically analyzed and normalized with the actin expression. Bardiagram in each panel shows normalized protein expressions in arbitraryunits. In bar diagram, the bars represent protein expressions in cellstreated with vehicle control (a), LI/DD-II/054A/01 (b), AR 933 (c) andcomposition 1B (d).

FIG. 10A: Bar diagrammatic representation of body mean weight gain inHFD induced metabolic syndrome model of SD rats supplemented without (1)or with (2) LI/DD-II/054A/01 from week-1 to week-8 of treatment. Eachbar represents mean±SD, *p <0.05.

FIG. 10B: Line diagrammatic representations of body weight in dietinduced metabolic syndrome model of SD rats supplemented with (2) orwithout (1) LI/DD-II/054A/01. Each line indicates change in mean bodyweight gain during eight-week treatment period.

FIG. 11: Bar diagrammatic representation of increase in serumadiponectin concentration in diet induced metabolic syndrome model ofSprague Dawley rats. Each bar indicates mean±SD of serum adiponectinconcentration at 0 day and after 56 days of treatment with eithervehicle (1) or LI/DD-II/054A/01 (2) as indicated in the diagram. N=6, *indicates statistical significance (t-test, 8 weeks vs. 0 week).

FIG. 12: Bar diagrammatic representation of reduction of HOMA Index inLI/DD-II/054A/01 supplemented metabolic syndrome model of Sprague Dawleyrats. Each bar indicates mean±SD of HOMA Index (arbitrary units) at 0week and at 8 weeks of supplement with either vehicle (1) or 250 mg/kgof LI/DD-II/054A/01 (2). N=6; * indicates statistical significance(t-test, LI/DD-II/054A/01 group vs. control at 8 weeks).

FIG. 13: Bar diagram representation of % reduction in body weight indiet induced obese model of Sprague Dawley rats. The bars 1 to 5represent % reductions in body weight in treatment groups supplementedwith LI/DD-II/054A/03 (100 mg/kg), LI/DD-II/054A/03 (250 mg/kg), AR 933(250 mg/kg), composition 1D (250 mg/kg) and sibutramine (7 mg/kg)respectively.

DETAILED DESCRIPTION OF THE INVENTION

Obesity is excess body weight for a particular age, sex and height as aconsequence of imbalance between energy intake and energy expenditure.The primary causes of obesity are either due to overeating, inadequateexercise or eating disorder, some genetic disorders, underlying illness(e.g. hypothyroidism), certain medications or sedentary lifestyle.Obesity increases the risk of many diseases and health conditions suchas hypertension, dyslipidemia (for example, high total cholesterol orhigh levels of triglycerides), type 2 diabetes, coronary heart disease,stroke, gallbladder disease, osteoarthritis, sleep disorders,respiratory problems, tumors (endometrial, breast, and colon),arteriosclerosis and heart failure.

Metabolic syndrome is a condition involving a set of disorders thatenhances the risk of heart disease. The major components of metabolicsyndrome are excess weight, the cardiovascular parameters (high bloodpressure, dyslipidemia, high levels of triglycerides and low levels ofHDL in the blood), atherosclerosis, diabetes and insulin resistance. Asubject suffering with several of these components, i. e. metabolicsyndrome is highly prone to heart disease, though each component is arisk factor. Adipocytes and macrophages play important role in thepathogenesis of metabolic syndrome and disease components associatedwith it. They both share a number of common features, including theability to phagocytize and kill microorganisms and to secrete cytokinessuch as tumor necrosis factor (TNF) and interleukin-1(IL-1). Criticaltranscription factors in adipocytes involved in regulating theexpression of cytokines, inflammatory molecules, and fatty acidtransporters are also expressed and have similar biologic roles inmacrophages. For example, activation of PPAR, a member of thenuclear-receptor super-family of ligand-activated transcription factors,is associated with differentiation of both types of cells. Inadipocytes, PPAR regulates adipocyte development and glucosehomeostasis. In macrophages, PPAR regulates expression of inflammatorygenes and is involved in the development of atherosclerotic lesions. Themacrophages over express Matrix Metalloproteinase-1 (MMP-1) and MatrixMetalloproteinase-3 (MMP-3) under certain disease conditions associatedwith metabolic syndrome. Similarly, the adipocytes in addition toaccumulating fat during the obesity development produce and circulateseveral low molecular weight bioactive protein molecules having powerfuleffects throughout the body. These protein markers are related todifferent components of metabolic syndrome. The expression andproduction of several of these metabolic markers, which include but notlimited to PPAR-γ, Adipose Differentiation Related Protein (ADRP), CD36,Adipocyte Fatty-Acid-Binding Protein (aP2/FABP4/A-FABP), Beta-3adrenergic receptor (β3-AR), adiponectin and Perilipin, become abnormalduring obesity and metabolic syndrome and other disease conditionsassociated with metabolic syndrome.

Atherosclerosis, also known as coronary heart disease (CHD), is one ofthe major vascular complication and important component of metabolicsyndrome that has enormous impact on the human health. It is a chronicinflammatory reaction to modified lipoproteins, primarily oxidized lowdensity lipoproteins (Ox LDL). Atherosclerosis is thought to develop asa result of lipid uptake by vascular-wall macrophages leading to thedevelopment of foam cells and the elaboration of cytokines andchemokines resulting in smooth muscle-cell proliferation (Berliner, J.A., Circulation, 91: 2488-2496, 1995, Boring, L., et. al., Nature, 394:894-897, 1998). Cluster of Differentiation 36 (CD36) protein has beenproven to play a key role in the process of atherosclerosis.

A brief description of some of the metabolic biomarker molecules,digestive enzymes and the metabolic processes that are involved in thepathogenesis and control of metabolic syndrome and the diseaseconditions associated is outlined below:

1. Matrix Metalloproteinases:

Matrix Metalloproteinases (MMPs) are zinc dependent endopeptidases, thatare capable of breaking down all kinds of extra cellular matrixproteins, such as collagen, that are normally found in the spacesbetween cells in tissues. MMPs are divided primarily into threeprincipal groups, the fibroblast collagenase-1 (MMP-1) formed of thecollagenases, the gelatinases comprising gelatinase A (MMP-2) and thegelatinase B (MMP-9), and the stromelysines comprising stromelysine-1(MMP-3) and matrilysine (MMP-7). An excess of metalloproteinase leads todegradation of biomolecules such as collagen, proteoglycon and gelatin,which can have fatal consequences on epidermis and can also generatediseases of the cartilages, inflammation etc.

MMPs are thought to participate in the pathogenesis of coronary arterydisease (CAD), particularly in the occurrence of acute coronary syndrome(ACS). Studies show that the expression and regulation of MMPs and theirtissue inhibitors (TIMPs) were evaluated in premature CAD. The plasmaconcentrations/activities of MMP-2, MMP-3 and MMP-9, TIMP-1, and TIMP-2in 80 patients (49 with ACSs and 31 with stable CAD) and 40 controlswere evaluated in clinical study and it was concluded that MMP and TIMPplasma levels in premature CAD are linked to clinical presentation andmarkers of inflammation and metabolic disorders rather than to geneticpolymorphisms. Similarly, macrophage-activity (monocyte chemoattractantprtein-1, neopterin), tissue-remodeling (matrix metalloproteinase-9) andthrombosis (tissue-factor) related biomarkers were found to beconsistently elevated in Acute Coronary Syndrome (ACS) compared tostable coronary artery disease (CAD)

The role of MMPs [both positive and negative] in obesity and in thedevelopment of adipose tissue has been investigated by severalinvestigators. A few are quoted below:

In a study the investigators studied the differential expression of MMPsand TIMPs by Northern blot and real-time PCR in two genetic models ofobesity (ob/ob and db/db mice) and in a diet-induced model of obesity(AKR mice). They have concluded that mRNA levels for MMP-2, MMP-3,MMP-12, MMP-14, MMP-19, and TIMP-1 are strongly induced in obese adiposetissues compared with lean tissues [Chavey C et al., J Biol Chem. 2003;278(14):11888-96].

In a similar study on nutritionally induced obesity mouse, theexpression of MMP-3, -11, -12, -13, and -14 and TIMP-1 mRNAs was foundto be upregulated when compared to those on the standard diet. It wasalso observed in an in vitro study that the adipogenesis was reduced inthe presence of a synthetic MMP inhibitor [Maquoi E et al., Diabetes.2002; 51(4):1093-101].

2. Peroxisome Proliferator-Activated Receptor (PPAR)-γ:

Peroxisome proliferator-activated receptor γ (PPAR γ) is a nuclearreceptor that plays a pivotal role in obesity and diabetes. An increasein adipose tissue mass can be the result of the production of new fatcells through the process of adipogenesis and the deposition ofincreased amounts of cytoplasmic triglyceride or lipid droplets percell. In the adipogenesis process, proliferation of preadipocytes orprecursor fat cells is followed by the differentiation of these cells tothe mature adipocyte phenotype. PPAR γ is expressed predominantly inadipose tissue, wherein it is known to play a critical role in adipocytedifferentiation and fat deposition.

The activation of PPAR γ on the other hand leads to an improvement ofinsulin action. PPAR γ is the molecular target of the thiazolidinedione(TZD) class of antidiabetic drugs, such as troglitazone (Rezulin),rosiglitazone (Avandia), and pioglitazone (Actos). Adipose tissuedevelopment and insulin sensitivity were found to be greatly impaired inPPAR γ2 knockout mice indicating its role in obesity and diabetes. PPARγ is a key regulator of fatty acid uptake and lipogenesis in addition toadipose cell differentiation through its influence on the production ofthe enzymes required for lipid storage and metabolism (Zhang, J.,Proceedings of National Academy Sciences 2004; 101; 10703-10708, 2004).

3. Adipose Differentiation Related Protein (ADRP):

ADRP is a 50 kD protein and it's mRNA (ADRP mRNA), which is 1.7 Kb insize, is expressed at high level in adipose tissue. The expression ofADRP is very low in undifferentiated adipocytes, but ADRP mRNA reaches50 to 100-fold in few hours after the onset of adipose differentiationprocess. ADRP is also found in many different types of cells and tissuesthat accumulate or synthesize lipids. The above thus indicate thepossible role of ADRP in the formation or stabilization of lipiddroplets in adipocytes and other cells. ADRP specifically enhancesuptake of long chain fatty acids by adipose tissue. Hence ADRP is animportant target to identify the compounds that can potentially controlobesity and diabetes through regulation of the expression of ADRP.

4. Adipocyte CD36:

CD36 is a common protein marker expressed by both adipocytes andmacrophages. The CD36 expressed in adipocytes is known to function as afatty acid transporter (FAT). Studies on adipocytes showed that CD36mRNA is a marker of adipocyte differentiation. It is a scavengerreceptor that binds and internalizes oxidized LDL (Ox LDL) inmacrophages. CD36 also functions as a long-chain fatty acid (LCFA)transporter to facilitate the uptake of LCFAs in adipocytes. The CD36expression is up-regulated by PPAR during the differentiation of bothtypes of cells. It is also shown that the adipocytes can endocytose andlysosomally degrade Ox LDL, mainly mediated by CD36. The CD36 nullanimals showed significant decrease in binding and uptake of oxidizedlow density lipoprotein and showed significant increase in fastinglevels of cholesterol, nonesterified free fatty acids, andtriacylglycerol.

5. Macrophage CD36:

CD36 is a prototypic member of the class B scavenger receptor family.The endogenous (e.g., macrophages, adipocytes, platelets, microvascularendothelial cells and specialized epithelial cells) and ectopic (e.g.,melanoma cells and fibroblasts) expression of this multi-ligand receptoron the surface of cells confers phagocytic activity for engulfment ofapoptotic cells. CD36 is widely expressed and may interact with multipleextracellular ligands including thrombospondin-1 (TSP-1), long chainfree fatty acids (FFAs), modified (oxidized) low-density lipoprotein(Ox-LDL), advanced glycation end (AGE) products, collagen I and collagenIV [PLoS Medicine, 2: 152-161, 2005]. CD36 is expressed on the surfaceof monocytes and macrophages and mediates uptake of oxidized low-densitylipoprotein (Ox-LDL) [Nozaki, S., J. Clin. Invest. 96: 1859-1865, 1995]as well as to play a role in diverse cellular processes including foamcell formation, fatty acid transport, engulfment and removal ofsenescent cells, suppression of angiogenesis, and cell-matrixinteractions. The CD36-dependent uptake of Ox-LDL has been shown to becritical to cholesterol accumulation and subsequent foam cell formation;activities that likely contribute to the observed involvement of CD36 inmouse models of atherogenesis [Michael E et al, J. Exp. Med., 203:2613-25, 2006].

CD36 may initiate atherosclerotic lesions and be an important riskfactor of cardiovascular disease. In mice lacking the CD36 receptor,foam-cell formation and vascular lesion development were indeedinterrupted [Febbraio M., et. al., J Clin Invest 105:1049-1056, 2000].Hyperglycemia-induced synthesis of CD36 in macrophages has beenassociated with increased uptake of Ox-LDL by macrophages and foam cellformation in atherosclerotic lesions in people with diabetes (PLoSMedicine, 2: 152-161, 2005]. The increased Peroxisomeproliferator-activated receptor-γ (PPAR-γ) obtained in response to highglucose levels lead to an increased expression of CD36 in macrophage andcontribute accelerated atherosclerosis.

The foregoing data thus demonstrate a correlation between increased CD36expression and hyperglycemia in atherosclerotic vascular lesions, whichthus offers potential opportunity and advantage to use CD36 as apotential molecular maker of atherosclerosis.

6. Leptin:

Leptin plays an important role in regulating energy expenditure inresponse to food intake. Leptin is an important adipocytokine of adiposetissues, which further contain low and medium molecular weight proteinslike adiponectin, tumor necrosis factor-alpha (TNF-alpha), interleukin-6(IL-6), resistin, plasminogen-activating inhibitor-I (PAI-1), andangiotensinogen. Together these cytokines play an important role in theadipose tissue physiology and are believed to be a link between obesity,insulin resistance and endothelial dysfunction.

In vivo gene expression of leptin, Fatty acid translocase (FAT/CD36),PPAR-gamma2, Uncoupling protein (UCP)-2, UCP-3, and TNF-alpha insubcutaneous adipose tissue is regulated by circulating lipidsindependent of insulin. Hence prolonged hyperlipidemia may contribute toincreased fat metabolism and storage as a result of the increasedexpression of these proteins. [Nisoli E et al.; Diabetes. 2000 March;49(3):319-24].

7. Oxidized LDL:

LDL cholesterol which is known as bad cholesterol becomes more dangerouswhen it is oxidized. Oxidized LDL can produce inflammation in arteriesthat supply blood to various organs and tissues. This leads toAtherosclerosis and increases the risk of heart attack or stroke.

Holvoet et al showed for the first time that the metabolic syndrome isassociated with a higher fraction of oxidized LDL and thus with higherlevels of circulating oxidized LDL. In this study, oxidized LDL wasmeasured with a monoclonal antibody-based enzyme-linked immunosorbentassay. They proved that increased concentration of oxidized LDL wasassociated with increased incidence of metabolic syndrome overall, aswell as its components of abdominal obesity, hyperglycemia, andhypertriglyceridemia. [Holvoet P et al., JAMA. 2008 May 21;299(19):2287-93.].

A study conducted on women with polycystic ovarian syndrome (PCOS)manifested that elevated OxLDL and a direct relation of ApoE(Apoliprotein) and nonesterified fatty acids with insulin resistancecould possibly have been the increase risk for premature atherosclerosisin these women.

8. Monocyte Chemotactic Protein (MCP-1):

Monocyte chemotactic protein-1 (MCP-1), a member of the small induciblegene (SIG) family, plays a role in the recruitment of monocytes to sitesof injury and infection. MCP-1 has also been called small induciblecytokine A2 (SCYA2) and monocyte chemotactic and activating factor(MCAF). MCP-1 has been found in the joints of people with rheumatoidarthritis, which may serve to recruit macrophages and perpetuate theinflammation in the joints. MCP-1 has also been found elevated in theurine of people with lupus as a warning sign for inflammation of thekidney. [http://www.medterms.com/script/main/art.asp?articlekey=33740].

Recently, MCP-1 has been reported to be a novel adipocytokine involvedin the development of obesity-associated insulin resistance andatherosclerosis. [Kawada T, et. al., Asia Pac J Clin Nutr. 2008; 17(1):126-30]. MCP-1 along with a number of other adipokines, includingleptin, adiponectin, tumour necrosis factor alpha, etc. is linked toinflammation and the inflammatory response. Obesity is characterized bya state of chronic mild inflammation, with raised circulating levels ofinflammatory markers and the expression. This elevated production ofinflammation-related adipokines is increasingly considered to beimportant in the development of diseases linked to obesity, particularlyType II diabetes and the metabolic syndrome. [Trayhurn P. and Wood I.S., Biochem Soc Trans. 2005; 33(Pt 5):1078-81].

9. Fatty-Acid-Binding Protein (aP2/FABP4):

FABPs are molecular chaperones linked to metabolic and inflammatorypathways. Different members of the FABP family exhibit unique patternsof tissue expression/distribution and are expressed most abundantly intissues involved in active lipid metabolism. FABPs play numerousfunctions. As lipid chaperones, for example, FABPs may activelyfacilitate the transport of lipids to specific compartments in the cell,such as to the lipid droplet for storage; to the endoplasmic reticulumfor signaling, trafficking and membrane synthesis; to the mitochondriaor peroxisome for oxidation [Masato, F et al, Nature Reviews/DrugDiscovery, Vol. 7: 489-503, 2008]. A-FABP is Fatty Acid Binding Proteinprominently expressed in mature adipocytes and macrophages. It is morefamiliarly known as FABP-4 and aP2. Adipocytes, however, expresssignificantly higher levels (approximately 10000-fold) of A-FABP thanmacrophages, upon their differentiation from pre-adipocytes andmonocytes respectively.

A-FABP is abundantly present in human serum and it may play a centralrole in the development of major components of the metabolic syndromesuch as obesity, type 2 diabetes and cardiovascular diseases, throughits distinct actions in adipocytes and macrophages and its ability tointegrate metabolic and inflammatory responses [Masato, F et al, NatureReviews/Drug Discovery, Vol. 7: 489-503, 2008]. The aP2 expressed inadipocytes regulates systemic glucose and lipid metabolism.

Blocking aP2 function is a novel approach to therapeutic strategy forthe treatment of obesity, tracking heart disease, Metabolic Syndrome andother components of Metabolic Syndrome.

10. β3-adrenergic receptor (β3AR):

The body's adrenergic system plays a major part in regulating energyexpenditure and lipolysis. In this process catecholamines mobilizeenergy-rich lipids by stimulating lipolysis in fat cells andthermogenesis in brown adipose tissue and skeletal muscle. The μ3AR isthe principal receptor mediating catecholamine-stimulated thermogenesisin brown adipose tissue, which in humans is distributed about the greatvessels in the thorax and abdomen [Thomas, G N, International Journal ofObesity, 545-551, 24, 2000]. The β3AR is also important in mediating thestimulation of lipolysis by catecholamines in the white fat cells ofseveral species, including humans. The brown adipose tissue differs fromwhite adipose tissue in that it has large numbers of mitochondriacontaining a so-called uncoupling protein, which can stimulate oxidativephosphorylation and thereby increase the metabolic rate. The role ofbrown adipose tissue is to oxidize lipids to produce heat and rid thebody of excess fat. White adipose tissue, which includes subcutaneousand visceral adipose tissue, is much more abundant. It serves to storefat, which can be mobilized by lipolysis to generate free fatty acidsfor use by other tissues.

Selective agonists of β3ARs are potentially useful in treating obesitybecause they could enhance energy expenditure with few β1- orβ2-adrenergic side effects. A number of μ3-adrenergic agonists have beendeveloped and tested experimentally. Hence the treatment withμ3-selective agonists can markedly increase energy expenditure anddecreases obesity.

11. Perilipin:

Perilipin is a protein that forms a coating around the lipid droplets inthe fat-storing cells in adipose tissue, called adipocytes. Perilipinacts as a protective coating against body's natural lipases, such ashormone-sensitive lipase, that break triglycerides into glycerol andfree fatty acids by a process called lipolysis.

In a study, it was suggested that the family of Perilipin [PLIN],adipophilin and TIP47 proteins may play key roles in obesity. PLIN is acandidate gene for obesity risk in humans as well as a modulator ofdietary response to therapies aimed to reduce body weight and risk ofmetabolic syndrome. [Tai E S et al; Curr Opin Lipidol. 2007; 18(2):152-6].

Following β-adrenergic receptor activation, protein kinase A (PKA)hyperphosphorylates perilipin localized at the surface of the lipiddroplet. Phosphorylated perilipin changes conformation and translocateaway from the lipid droplet, exposing the stored lipids tohormone-sensitive lipase-mediated hydrolysis of triglycerides(lipolysis) to release nonesterified fatty acids (NEFA). Perilipin isthus an important regulator of lipid storage, lipolysis and energybalance. Perilipin expression is elevated in obese animals and humans.Studies manifested a significant positive relationship between perilipinexpression and obesity (P<0.01, perilipin mRNA vs. percent body fat).Because of the potential importance of adipocyte lipolysis to obesityand insulin resistance, perilipin is an important target for developinganti-obesity drugs. Agents that inactivate or inhibit perilipin may findapplication as potential anti-obesity medications.

12. Adiponectin:

Adiponectin is an important adipokine and it was proved that low levelsof adiponectin are associated with disease states such as obesity,diabetes and cardiovascular disease. Administration of adiponectin wasproved to be beneficial in animal models of diabetes, obesity andatherosclerosis.

Adiponectin level in blood is decreased in obesity and it is consideredto have antidiabetic and antiatherogenic effect, whereas increasedleptin level in blood in obesity is associated with regulation ofappetite, energy expenditure, lipids and carbohydrates metabolism,cellular differentiation. In a study carried out on 80 patients (43female and 37 male) from Obese families, the fasting level of leptin(Elisa), adiponectin (Elisa) and von Willebrand factor (Elisa)lipidogram were performed. It was found that the leptin to adiponectinratio (Lep/AdipoR) in the blood was significantly higher in obesepatients in comparison to people with normal BMI. Relative OperatingCharacteristic (ROC) showed that with reference to obesity Lep/AdipoRhad the highest discriminatory value. The estimation of Lep/AdipoR canbe used as additional index in evaluation of obesity complications suchas insulin resistance and endothelial dysfunction.

It was also proved that high plasma concentrations of adiponectin areassociated with lower risk of Myocardial Infarction in men. [Pischon Tet al., JAMA. 2004 Apr. 14; 291(14):1730-7]. Hence the phytochemicalextracts or fractions or compounds that enhance the adiponectin levelscan have beneficial effects on obesity, diabetes, cardiovascular systemand metabolic syndrome and other disease components associated withmetabolic syndrome.

13. Protein Tyrosine Phosphatase 1B (PTP-1B):

Resistance to the hormone insulin is the hallmark of type 2 diabetes andobesity. Protein tyrosine phosphatase 1B (PTP-1B) is regarded as anegative regulator of insulin signal transduction in insulin sensitivecells such as adipocytes, muscle cells and hepatocytes. In insulinresistant diabetes and obesity, the PTB-1B is over expressed and itsenzyme activity is increased. Over expression of PTP1B decreases insulinreceptor and IRS-1 Phosphorylation and thus produces insulin resistance(Theodore O. J., et al., Nature Reviews Drug Discovery, 1; 696-709,2002; Carol L. V., et. al., J. Biol. Chem. 275: 18318-18326, 2000.).Therefore, agent(s) providing PTP-1B inhibition has become an emergingtherapeutic promise to patients with at-risk obesity and or type-2diabetes.

14. Adipogenesis:

Adipogenesis is the differentiation and proliferation of pre-adipocytesinto major adipocytes or fat cells and it has been one of the mostintensely studied models of cellular differentiation. In theadipogenesis process, proliferation of preadipocytes or precursor fatcells is followed by the differentiation of these cells to the matureadipocyte phenotype. The nuclear receptor PPAR γ is expressedpredominantly in adipose tissue, where it is known to play a criticalrole in adipocyte differentiation and fat deposition. Many drugs in themarket for the treatment of diabetes type-II, however involve overexpression of PPAR γ and promotion of adipogenesis.

Adipocytes secrete proteins exhibiting either beneficial (leptin,adiponectin) or deleterious effects (angiotensinogen). A disturbance inthe balance between these various secreted factors, in association withthe effect of secretory products from macrophages (cytokines), leads tothe development of metabolic syndrome.

15. Lipolysis:

Lipolysis is the breakdown of stored lipid in adipocytes.β3-Adrenoreceptor agonists can stimulate lipolysis in the white adiposetissue and thermogenesis in the brown adipose tissue. The phytochemicalagents having the lipolysis activity could be useful in the treatment ofobesity, metabolic syndrome and other metabolic disorders. Adiposetissue lipolysis is the catabolic process leading to the breakdown oftriglycerides stored in fat cells and release of fatty acids andglycerol. The proteins involved in the lipolytic process constitute drugtargets for the treatment of obesity and the metabolic syndrome.

16. Alpha-Amylase (α-Amylase):

α-amylase is an enzyme that converts complex carbohydrates such asstarch into sugar. Starches cannot be absorbed unless they are firstbroken down by the digestive enzyme amylase and other secondary enzymes.When carbohydrates are consumed, enzymes in the digestive tract breakthese large molecules down into smaller sugar molecules, which areabsorbed through the intestine. Recently, starch blockers have beenshown to be effective treatments for the control of obesity. Amylaseinhibitors thus have gastrointestinal and metabolic effects that may aidin the treatment of diabetes and obesity.

Plants also use α-amylase inhibitors as a defense strategy. Theseinhibitors impede the digestive action of α-amylases and proteinases inthe insect gut, thereby acting as insect anti-feedants. Thealpha-amylase inhibitors isolated from the plants may be used as starchblockers to considerably reduce the quantity of glucose originating fromthe starches present in the diet, and reduces the appetite afterrepeated administration.

17. Alpha-Glucosidase (α-Glucosidase):

Alpha-glucosidase is an enzyme that catalyses the degradation of complexcarbohydrates into glucose. As such its action is similar to α-amylase.By inhibiting this enzyme, carbohydrates are not broken down asefficiently and glucose absorption from intestine is delayed, resultingin a slower and lower rise in blood glucose throughout the day,especially right after meals. A few α-glucosidase inhibitors, such asacarbose, miglitol and voglibose are oral anti-diabetic drugs commercialavailable in the market for the treatment and control of diabetesmellitus type 2. Plant extracts, fractions or pure compounds havingα-glucosidase inhibition may thus be useful to establish greaterglycemic control over hyperglycemia in diabetes mellitus type 2,particularly with regard to postprandial hyperglycemia.

Metabolic Syndrome is recognized as an important disease which can besingle or can be a set of diseased conditions, such as obesity, diabetesand atherosclerosis and if it is left untreated leads to severalcomplications. Even though several classes of drugs are available in themarket for the treatment of different components of Metabolic Syndromeand many of them are associated with a number of side effects, very fewmedicines are available to treat Metabolic Syndrome and none of them arecomprehensive in addressing all the associated diseases. Hence thereexists a great medicinal need for developing the protection andtreatment against metabolic syndrome, obesity, diabetes andatherosclerosis especially using safe and beneficial natural compounds.

One of the key developments in obesity research in the past decades hasbeen the general recognition that obesity is a chronic low levelinflammation. The link between obesity and inflammation has been obviousfrom the increased plasma levels of several inflammatory markersincluding cytokines (TNFα, IL-6) and acute phase proteins likeC-reactive protein (CRP) in obese individuals (Stienstra R., et. al.,2007, article ID 95974). Thus obesity, diabetes and atherosclerosis aswell as other components of the metabolic syndrome have been casuallylinked to inflammation. It has also been theorized in recent years thatchronic, low-grade tissue inflammation related to obesity contributes toinsulin resistance, the major cause of Type 2 diabetes (Science News,Science Daily, U.S., Nov. 7, 2007).

The research activity in the area of metabolic disorders has been a highpriority target for numerous scientists around the world, with a specialinterest in finding alternative solutions, especially those based onproducts of plant origin, as the plant derived products are consideredto be natural and safe, in contrast to the commercial drugs of syntheticorigin. Keeping this in mind and in conjunction with the urgent need forthe prevention, control and treatment of metabolic syndrome, obesity,diabetes, atherosclerosis and endothelial dysfunction and othermetabolic disorders, the inventors have conducted extensive researchinvestigation involving several in vitro and in vivo experiments onseveral plant extracts, fractions and pure compounds and accidentallyfound that administration of the extracts, fractions, active compoundsderived from the herb Sphaeranthus indicus in a therapeuticallyeffective amount in cell based studies potently ameliorated metabolicprocesses which include inhibition of adipogenesis and also promotion ofadipolysis (lipolysis). The inventors also found unexpectedly that theadministration of one or more of the ingredient(s) selected from theextracts, fractions, active compounds derived from the herb Sphaeranthusindicus in a therapeutically effective amount in cell based studiespotently ameliorated the levels of certain biomarker molecules orbiological proteins that are altered during metabolic syndrome, obesity,diabetes, atherosclerosis, endothelial dysfunction and other diseaseconditions associated with metabolic syndrome.

The inhibition of lipid accumulation in differentiated adipocytesexhibited by the ethyl actate extract (LI/DD-II/054A/01) of the flowerheads of Sphaeranthus indicus was assessed in 3T3-L1 mouse pre-adipocytecells. The inhibition of fat accumulation in the treated cells wascompared with the mock treated differentiated adipocytes and percentageinhibition was measured. Unexpectedly, the ethyl acetate extract(LI/DD-II/054A/01) of Sphaeranthus indicus significantly inhibited theadipogenesis 65.9% inhibition of lipid accumulation at 10 μg/ml assummarized in Table 1.

Similarly, the pro-lipolytic activity of the ethyl actate extract(LI/DD-II/054A/01) of Sphaeranthus indicus was assessed indifferentiated/mature adipocytes using 3T3-L1 pre-adipocyte cells. Thelipolytic activity was assessed in mature adipocytes by measuring freeglycerol secreted into the culture medium as per the procedure ofAdipolysis Assay Kit (Chemicon International, USA). The percentageincrease in glycerol concentration in the sample solutions compared tothe control containing the known concentrations of glycerol correspondsto the percentage acceleration of lipolysis exhibited by extractLI/DD-II/054A/01 of Sphaeranthus indicus. Unexpectedly, LI/DD-II/054A/01showed moderate enhancement of lipolysis/adipolysis with 26.7% increasein lipolysis at 25 μg/ml as summarized in Table 2.

Bio-assay guided purification was undertaken on LI/DD-II/054A/01 toidentify the active compound and it was found accidentally that7-α-hydroxy-4,11(13)-eudesmadien-12,6-olide also known as7-hydroxyfrullanolide (LI054A01; 1) was found to be responsible for theactivity. The active compound7-α-hydroxy-4,11(13)-eudesmadien-12,6-olide also known as7-hydroxyfrullanolide (LI054A01) exhibited superior activity and showed68.7% inhibition of lipid accumulation (adipogenesis) at 0.5 μg/mlconcentration and 47.8% increase lipolysis at 5 μg/ml concentration assummarized in Tables 1 and 2.

It was also found surprisingly that the ethyl actate extract(LI/DD-II/054A/01) of Sphaeranthus indicus possessed potent MatrixMetalloproteinase-1 (MMP-1) inhibition activity (FIG. 1) in PMA inducedA2058 human melanoma cells. Phorbol myristate acetate (PMA) is a potentknown intracellular oxidative stress-inducing agent. In cardiovascularmanifestation, such as atherosclerosis, over production of oxidativestress markers is a critical factor. In addition, LI/DD-II/054A/01 alsoshowed potent Matrix Metalloproteinase-3 (MMP-3) inhibition (FIG. 2) inInterleukin-1β induced A549 human lung tumor cells. In atherosclerosis,over production of pro-inflammatory proteases such as MMP-3, MMP-9,MMP-13 under the influence of pro-inflammatory cytokines such as IL-1β,is important crucial for development and progression of atheroscleroticlesions and aneurysm formation

The inventors have then evaluated the modulation of metabolic biomarkersthose are primarily responsible for the adipogenesis processes, insulinresistance in type 2 diabetes, obesity, metabolic syndrome and othermetabolic disorders such as Peroxisome proliferator-activated receptorgamma (PPARγ), CD36, adipocyte fatty acid binding protein 4 (FABP4 oraP2), Perilipin, and beta-3 Adrenergic Receptor (β3AR) duringadipogenesis process in 3T3-L1 adipocytes by ethyl actate extract(LI/DD-II/054A/01) of Sphaeranthus indicus and 7-hydroxyfrullanolide(LI054A01) using an immunoblot assay. The mouse pre-adipocyte 3T3-L1cells under maintenance in Dulbecco's Modified Eagle's Medium (DMEM)were pre-treated with different concentrations of LI/DD-II/054A/01 for 2h, followed by addition of differentiation medium containing 500 nMinsulin, 1.0 μM dexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX)for 48 h. Thereafter, cells were further incubated with postdifferentiation medium (DMEM containing 100 nM insulin) in presence orabsence of LI/DD-II/054A/01 or LI054A01 for further 8 days. Finally, thecells were processed and lysed with the lysis buffer. The proteinextracts were evaluated by immunoblot assay, and the immuno-reactivebands were developed with West-pico chemiluminescent substrate and theblot images were captured in a Kodak Image Station, normalized withexpression of actin.

It was found surprisingly that both the ethyl acetate extract(LI/DD-II/054A/01) and the active compound 7-hydroxyfrullanolide(LI054A01) potently ameliorated the levels of several adipocytedifferentiation markers such as Peroxisome proliferator-activatedreceptor gamma (PPARγ), CD36, Fatty Acid Binding Protein 4 (aP2/FABP4)and intracellular lipid droplet surface associated protein (perilipin)(FIG. 3) in a dose dependent manner. The down regulation of severalmarker proteins in LI/DD-II/054A/01 treated adipocytes suggests that theethyl acetate extract of Sphaeranthus indicus exerts multiple beneficialroles in controlling the adipogenic differentiation process; by (1)inhibiting cellular differentiation by down regulating PPARγ, which is anuclear receptor protein that functions as a transcription factor forregulation of cellular differentiation, development and metabolism. (2)restricting cholesterol ester uptake by inhibiting CD36, which is aclass B scavenger receptor involved in lipid uptake, (3) decreasingintracellular adiposity and intracellular lipid transport by reducingFABP4/aP2 level, which acts as a transport protein for long chain fattyacids. Moreover, down regulation of perilipin protein inLI/DD-II/054A/01 treated adipocytes strongly indicate the reduced fatstore in the cytoplasm. Perilipin is a protein that coats lipid dropletsin adipocytes. It offers protection from the action of hormone-sensitivelipase, which breaks triglycerides into glycerol and free fatty acidsfor use in metabolism or lipolysis. Therefore it is indicative thatethyl acetate extract of Sphaeranthus indicus provides such a statewhere the stored lipids are more susceptible to enzymatic break downinto glycerol and free fatty acids by thinning the perilipin coat aroundthe lipid filled vesicles.

In addition, the beta-3 Adrenergic Receptor (β3AR) expression/productionin 3T3-L1 adipocytes was significantly enhanced by LI/DD-II/054A/01 in adose dependent manner as shown in FIG. 3. This is indicative of weightloss through increasing energy expenditure via increasing intracellularcAMP and activation of the mitochondrial uncoupling protein 1 in theadipose tissue.

In macrophages, CD36 is a scavenger receptor that mediates uptake ofoxidized low-density lipoprotein (OxLDL) and subsequent foam-celldevelopment. Therefore, increased level of CD36 in macrophages has beenconsidered as a predictive marker for development of atherosclerosis.Inhibition of CD36 protein expression in high glucose induced J774macrophage cells in presence or absence of LI/DD-II/054A/01 and LI054A01was evaluated using immunoblot assay. Briefly, equal amount of celllysates protein obtained after treating the cells with LI/DD-II/054A/01or LI054A01 was resolved in 7.5% SDS-PAGE; thereafter, the proteins weretransferred to nitrocellulose membrane. After blocking the non-specificsites, the membrane was incubated with CD36 antibody (R&D Systems,Minneapolis, Minn.). Finally, the specific immuno-reactive bands weredeveloped with West-pico chemiluminescent substrate (PierceBiotechnology, IL, USA), and the immunoblot images were recorded in aKodak Image Station (Kodak, USA). Band intensities were calculateddensitometrically and normalized with expression of actin in respectivesamples. The results are summarized in FIG. 3. The representativeimmunoblot image indicated that LI/DD-II/054A/01 dose dependentlyinhibited the CD36 protein expression in high glucose induced J774macrophage cells. The active compound LI054A01 however has shown 10times better inhibition of CD36 compared to that shown byLI/DD-II/054A/01. This unexpected observation also provides support infavor of anti-atherosclerotic properties of the extracts and compoundsof Sphaeranthus indicus.

It is also interesting to note that the ethyl acetate extract(LI/DD-II/054A/01) also potently inhibited Monocyte Chemotacticprotein-1 (MCP-1) and oxidized LDL in high glucose induced macrophagecells. These markers are critically important for differentiation intofoam cells to develop atherosclerotic plaque and are considered aspotential biological markers for development and progression ofatherosclerosis (FIG. 7).

Similarly, the modulation of adiponectin protein by ethyl acetateextract (LI/DD-II/054A/01) of Sphaeranthus indicus in 3T3-L1 adipocyteswas evaluated in Western immunoblot assay. The cell culture, treatmentprotocol and immunoblot assay methodology were as per the standardprotocol and as briefly described above for metabolic markers. Theextract LI/DD-II/054A/01 also showed moderate upregulation ofadiponectin protein expression in 3T3-L1 mature adipocytes as depictedin FIG. 8. Adiponectin is a hormone secreted by adipocytes. It reducesintracellular triglyceride content and up-regulates glucose uptake bypotentiating insulin signaling, thus it provides protection from bothadipogenicity and from developing insulin resistant diabetes or type 2diabetes. Therefore, our finding indicates that the extracts ofSphaeranthus indicus provides protection against developing obesity,insulin resistant or Type 2 diabetes and also helps in attenuatingendothelial dysfunction disorders as well. These extracts can thus beuseful in the prevention, treatment and control of above metabolicdisorders.

The effect of Sphaeranthus indicus ethyl acetate extract(LI/DD-II/054A/01) on insulin sensitivity was evaluated by studying themodulation of Protein Tyrosine Phosphatase-1B (PTP-1B) activity in3T3-L1 preadipocytes by LI/DD-II/054A/01. The 3T3-L1 preadipocytes werecultured in Dulbecco's Modified Eagle's Medium (DMEM) as per standardprotocol and treated with LI/DD-II/054A/01 for 48 h. The cells werelysed with cell lysis buffer and the cell lysates were clarified at14,000×g for 5 min at 4° C. The PTP-1B activity was estimated by usingsubstrate reagent containing 10 mM 4-nitrophenylphosphate (pNPP, SigmaChemical Co., MO, USA) following the standard protocol and the colorreaction was read in a microplate ELISA reader (BioRad, USA). Theresults showed unexpectedly that LI/DD-II/054A/01 could be a potentinhibitor of PTP-1B activity in 3T3-L1 preadipocytes (FIG. 9).

Protein-tyrosine phosphatase (PTP)-1B acts as a physiological negativeregulator of insulin signaling by dephosphorylating the phosphotyrosineresidues of the insulin receptor and Insulin receptor-substrate complex1 (IRS-1). Silencing of PTP-1B gene in a prior animal studyastonishingly provided resistance from developing type 2 diabetes.Therefore, inhibition of PTP-1B has recently been emerged as a potentialtarget to treat type 2 diabetes. Interestingly, LI/DD-II/054A/01 in thepresent invention exhibited significant inhibition of PTP-1B activity inadipocytes (FIG. 9). This observation thus indicates that the extractsof Sphaeranthus indicus can also be used as a potential therapeuticintervention to treat type 2 diabetes.

It was quite unexpected and surprising to see that a single ingredientderived from Sphaeranthus indicus could be able to modulate the markerproteins related to many disease conditions associated with metabolicsyndrome and other metabolic disorders. This unexpected result suggeststhat Sphaeranthus indicus derived extract(s), fraction(s) andcompound(s) could be a potential therapeutic agent to prevent, treat andcontrol metabolic syndrome, obesity, diabetes, atherosclerosis,endothelial dysfunction, chronic kidney disease (CKD) and othermetabolic disorders in animals and humans.

The potent anti-metabolic syndrome effect shown by LI/DD-II/054A/01 invitro models was further evaluated in an in vivo model of metabolicsyndrome. Metabolic syndrome condition was experimentally induced inmale Sprague Dawley rats by feeding the rats with high fat, highcholesterol, high salt and high sucrose diet for eight weeks. Aftereight weeks of induction period, the rats were randomly divided into twogroups with six animals in each group and the treatment group animalswere supplemented orally with 250 mg/kg body weight of LI/DD-II/054A/01in 10 mL of 0.5% CMC in water for further 8 weeks. The control group ofanimals received only the vehicle (10 mL/kg of 0.5% CMC in water) duringthis period. Body weight of individual animal was recorded weekly forthe entire duration of the study. Mean body weights for the treatmentgroup and control group were determined. The body weight gain wascalculated at the end of 1st week, 4th week and 8th week afterinitiation of treatment in comparison to initial body weight.LI/DD-II/054A/01 at a dose of 250 mg/kg exhibited highly potent andstatistically significant (p<0.01) reduction in body weight gain(66.04%) in comparison to control group. The results of body weight gainin the treatment group and control group are summarized in FIGS. 10A and10B.

Assessment of serum adiponectin: Adiponectin is a protein hormoneexclusively secreted from the adipose tissue, which modulates number ofmetabolic processes including glucose homeostasis and lipid metabolism.The circulatory adiponectin concentration is inversely correlated withbody fat. Low level of adiponectin is related with obesity,cardiovascular disorder and insulin resistance. Therefore, this proteinhormone has been established as a promising marker of metabolic syndromeand disease conditions associated with metabolic syndrome. The serumadiponectin concentration in the treatment and control groups of animalswas assessed using double antibody based sandwich rat adiponectin ELISAkit. The data revealed that daily supplementation of LI/DD-II/054A/01 at250 mg/kg body weight for 8 weeks resulted in significant (p=0.00618)improvement in serum adiponectin concentration, when compared to thebaseline as summarized in FIG. 11. The control group, however, did notshow improvement in serum adiponectin concentration. HenceLI/DD-II/054A/01 has potential benefit in alleviating the symptoms suchas obesity, cardiovascular disorders, insulin resistant type-IIdiabetes, metabolic syndrome and other related disorders of metabolicsyndrome.

Supplementation of LI/DD-II/054A/01 at 250 mg/kg resulted in improvementin fat profile with 15.3, 12.7 and 22.9 percentage reductionsrespectively in serum cholesterol, LDL and triglycerides. This is wellcorroborated with its efficacy observed in improvement of adiponectinlevels.

Homeostasis Model Assessment (HOMA):

The HOMA index was calculated based on serum insulin and glucose levelsusing the formula: Fasting insulin concentration (μU/mL)×Fasting glucoseconcentration (mmol/L)/22.5. The data presented in FIG. 12 manifestedthat compared to the control group, supplementation of a daily dose of250 mg/kg of LI/DD-II/054A/01 for 8-weeks resulted in significantreduction of HOMA index. The Homeostatic Model Assessment (HOMA) iswidely considered as a measure of insulin resistance and beta cellfunction in clinical research. The data indicates that LI/DD-II/054A/01can be a therapeutic agent to improve insulin sensitivity and μ-cellfunction.

Based on the present animal study, it is obvious that LI/DD-II/054A/01not only reduces obesity but also ameliorates various symptoms of themetabolic syndrome including body weight gain, visceral and organ fatdeposition and improves lipid profile, glucose homeostasis, μ-cellfunction etc. The reduction in body weight gain obtained byLI/DD-II/054A/01 treatment was statistically significant. Additionally,fat tissue weight was also significantly reduced in LI/DD-II/054A/01treatment group. In conclusion, LI/DD-II/054A/01 can be effective agentfor the treatment of obesity, metabolic syndrome and other relatedmetabolic disorders.

Further, random screening studies have shown surprisingly that themethanol extract of Garcinia mangostana (AR 933) rind possesses potentα-amylase and α-glucosidase inhibitory activities in enzymatic studiesand exhibited potent anti-adipogenesis activity in cell based in vitroassays in 3T3-L1 cells. The most active compounds were found to beα-mangostin (2) and γ-mangostin (3) during the bio-assay guidedseparation of the methanol extract. The methanol extract comprisingα-mangostin and/or γ-mangostin also showed potent pro-lipolytic andanti-adipogenesis activity. The methanol extract also significantlyameliorated the levels of various key biomarker molecules or biologicalproteins those are primarily responsible for the adipogenesis and lipidbreak down processes, insulin resistance in type 2 diabetes, inmetabolic disorders and disease conditions associated with metabolicsyndrome. These include PPARγ, ADRP, adipocyte CD36, aP2/FABP4/A-FABP,β3AR, adiponectin, Perilipin and PTP-1B (FIGS. 5, 8 and 9). It is alsointeresting to note that AR 933 also potently inhibited the macrophageCD36, MCP-1, and oxidized LDL in high glucose induced macrophage cells(FIG. 8). Altogether, These suggest that AR 933 can also be used as apotential candidate to treat obesity, cardiovascular manifestations suchas atherosclerosis, metabolic syndrome and other metabolic disorders.

The inventors have then randomly prepared and tested severalcompositions comprising the extracts of Sphaeranthus indicus incombination with several plant extracts in an effort to find acomposition that can show better efficacy in ameliorating the metabolicprocesses and in the expression or production of biomarker moleculesassociated with metabolic disorders, and to identify a better agent forcontrolling, treating and preventing obesity, metabolic syndrome anddisease conditions associated with metabolic syndrome and metabolicdisorders. It was found accidentally that compositions comprising atleast one phytochemical component selected from the extract(s) orfractions or enriched fraction(s) or compound(s) derived fromSphaeranthus indicus in combination with atleast one phytochemicalcomponent selected from extracts or enriched fractions or compoundsderived from Garcinia mangostana can be very effective in theamelioration of certain metabolic processes and modulation of someimportant metabolic biomarkers involved in the metabolic processes.

The metabolic processes controlled by the above compositions includeanti-adipogenesis and pro-lipolysis, and α-amylase and α-glucosidaseenzyme inhibitions. The biomarkers ameliorated by the above compositionsinclude but not limited to MMP-1, MMP-3, PPARγ, ADRP, adipocyte CD36,macrophage CD36, MCP-1, Ox-LDL, aP2/FABP4/A-FABP, β3AR, adiponectin,Perilipin and PTP-1B.

More importantly, it was further observed accidentally that there was asynergistic inhibitory effect between the extracts of these two herbs inthe amelioration of certain metabolic processes and modulation of someimportant metabolic biomarkers involved in the metabolic processes whenthey are combined rather than using the individual constituentsseparately. The composition 1B, for example, obtained by combining unitdoses of ethyl acetate extract (LI/DD-II/054A/01) and Garciniamangostana methanol extract (AR 933) in 1:3 ratio showed synergisticinhibition of selected biomarker proteins such as ADRP, aP2, perilipinand PTP-1B in adipocyte 3T3-L1 cells and CD36, MCP-1 and Ox-LDL in mousemacrophage cells as shown in FIGS. 6, 7 and 9. In addition, composition1B also showed synergistic enhancement of the production of adiponectinin adipocyte cells (FIG. 8). The efficacy shown by the composition isbetter than the effects exhibited by each of the individual ingredients.

The potent anti-obesity properties and synergistic effects shown by theextracts of Sphaeranthus indicus and Garcinia mangostana and theircompositions in in vitro models were further evaluated in an in vivomodel of obesity. Obesity was induced in male Sprague Daley rats bysupplementing the rats with High Fat diet for eight weeks. After eightweeks of induction period, the rats were randomly allocated to variousgroups with seven animals in each group and the animals belonged to thetreatment groups were orally supplemented daily with 100 or 250 mg/kgbody weight of methanol extract of Sphaeranthus indicus(LI/DD-II/054A/03) or 250 mg/kg body weight of AR 933 or 250 mg/kg bodyweight of composition 1D containing LI/DD-II/054A/03 and AR 933 in 3:1ratio, each in 10 mL of 0.5% CMC in water for further 8 weeks. Thecontrol group of animals received only the vehicle (10 mL of 0.5% CMC inwater). Body weight of individual animal was recorded weekly, and meanbody weight of the animals in each group was determined. The body weightgain was calculated at the end of 1st week, 4th week and 8th week afterinitiation of treatment in comparison to respective initial body weight.LI/DD-II/054A/03 dose dependently inhibited the body weight gain intreatment group of rats. The rats supplemented with 100 mg/kg bodyweight of LI/DD-II/054A/03 exhibited 46.3% reduction in body weight gainin comparison with the control animals. Similarly, AR 933 andLI/DD-II/054A/03 at a daily dose of 250 mg/kg exhibited 41.3% and 80.1%reductions in body weight gain respectively. Interestingly, thecomposition 1D at the same dose level (250 mg/kg) exhibited more potentand significant reduction in body weight gain (89%) compared to thevehicle treated control group. The results of body weight gain for thetreatment groups and control group are summarized in FIG. 13.

From the foregoing, it is obvious that the reduction in weight gainshown by the composition 1D comprising Sphaeranthus indicus methanolextract (LI/DD-II/054A/03) and methanol extract of Garcinia mangostana(AR 933) in 3:1 ratio is better than the effect shown by the individualingredients LI/DD-II/054A/03 and AR 933, manifesting a synergisticeffect between these two ingredients.

Even though a few selected extracts have been used in this study, thisinvention covers all extracts, active fractions and active compounds ofthe Sphaeranthus indicus, which comprises the active ingredient7-hydroxyfrullanolide or other active ingredient(s) in the range of 0.1%to 99.9%. Preferably any organic solvent extract of Sphaeranthus indicusor a fraction or pure compound derived from the extract with or withoutstandardization to 7-hydroxyfrullanolide can be used. The medium forobtaining active extract may be selected from either organic solvents orwater or mixtures of organic solvent and water, preferably an organicsolvent. The list of organic solvents include but not limited to hexane,dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol,n-butanol, iso-propanol, methyl isobutyl ketone etc or the mixturesthereof. The crude extracts may be used as they are as a medicament orfor making the compositions. Alternatively, the extracts may be enrichedto any designated concentration of 7-hydroxyfrullanolide or one or moreactive ingredients in the range of 0.1% to 99.9% using solventpartitions or washings or column chromatography on silica or reversedphase silica or resin column using organic or aqueous solvents ormixtures thereof or crystallizations or combinations thereof prior tousing them directly for desired health application of the invention orfor making the compositions.

Similarly, methanol extract of Garcinia mangostana has been used todemonstrate the present invention. However, any organic solvent extractextract or mixed organic solvent extract or water or an extract obtainedby an extraction with a mixed solvent comprising water and watermiscible organic solvent and can also be used.

The present novel and inventory compositions comprise different aspectsof the invention cited below:

The word “component” widely used in the specification and claims of thepresent invention refers to herb powders, extracts, fractions, enrichedfractions, active compounds or phytochemicals and phytochemical actives.The word “component” is used in the description from now onwards as asubstitute to these terms.

The extract(s), fraction(s), active compound(s), phytochemicals ormixtures thereof derived from Sphaeranthus indicus can be used aspharmaceutical/dietary supplement/food ingredients for the prevention,treatment and control of obesity, diabetes, atherosclerosis, metabolicsyndrome and other metabolic disorders.

The pharmaceutical/dietary supplement/food ingredients mentioned in thepresent invention refers to the extract(s), fraction(s), activecompound(s), phytochemicals or mixtures thereof derived fromSphaeranthus indicus.

In an important aspect, the invention provides pharmaceutical/dietarysupplement/food ingredient composition(s) comprising at least onecomponent selected from the extract(s), fraction(s), active compound(s)and phytochemical(s) or mixtures thereof derived from Sphaeranthusindicus and at least one component selected from the extract(s),fraction(s), active compound(s) and phytochemicals or mixtures thereofderived from Garcinia mangostana, optionally comprising one or moreselected from biologically active components derived from plants,animals and microorganisms, pharmaceutically or dietetically acceptableactive ingredients, vitamins, minerals, vehicles, carriers and diluentsor mixtures thereof.

In the other important aspect the invention provides,pharmaceutical/dietary supplement/food ingredient(s) comprising atleastone component selected from the extract(s) or fraction(s) or activecompound(s) or phytochemicals or mixtures thereof derived fromSphaeranthus indicus for the control, prevention and treatment of one ormore disease conditions selected from but not limited to obesity, weightloss, diabetes, atherosclerosis, arteriosclerosis, cardiovasculardiseases, neurological disorders, Alzheimer's, cognitive disorders,oxidative stress, skin disorders, aging of the skin, UV irradiateddamage, hypertension, hypercholesteremia (LDL, HDL, VLDL),hyperlipidemia (triglycerides), immune deficiency, cancer, metabolicsyndrome and other metabolic disorders.

In another aspect the invention provides the pharmaceutical/dietarysupplement/food ingredient compositions comprising atleast one componentselected from the extract(s), fraction(s), active compound(s),phytochemical(s) or mixtures thereof derived from Sphaeranthus indicusin combination with one or more selected from biologically activecomponents derived from plants, animals and microorganisms,pharmaceutically or dietetically acceptable active ingredients,vitamins, minerals, vehicles, carriers and diluents or mixtures thereoffor the control, prevention and treatment of one or more diseaseconditions selected from but not limited to obesity, weight loss,diabetes, atherosclerosis, arteriosclerosis, cardiovascular diseases,neurological disorders, Alzheimer's, cognitive disorders, oxidativestress, skin disorders, aging of the skin, UV irradiated damage,hypertension, hypercholesteremia (LDL, HDL, VLDL), hyperlipidemia(triglycerides), immune deficiency, cancer, metabolic syndrome and othermetabolic disorders.

In another aspect, the invention provides the pharmaceutical/dietarysupplement/food ingredient compositions comprising at least onecomponent selected from the extract(s), fraction(s) and activecompound(s), phytochemical(s) or mixtures thereof derived fromSphaeranthus indicus and at least one component selected from theextract(s), fraction(s) and active compound(s), phytochemical(s) ormixtures thereof derived from Garcinia mangostana, optionally containingone or more selected from biologically active components derived fromplants, animals and microorganisms, pharmaceutically or dieteticallyacceptable active ingredients, vitamins, minerals, vehicles, carriersand diluents or mixtures thereof for the control and prevention andtreatment of one or more disease conditions selected from but notlimited to obesity, weight loss, diabetes, atherosclerosis,arteriosclerosis, cardiovascular diseases, neurological disorders,Alzheimer's, cognitive disorders, oxidative stress, skin disorders,aging of the skin, UV irradiated damage, hypertension,hypercholesteremia (LDL, HDL, VLDL), hyperlipidemia (triglycerides),immune deficiency, cancer, metabolic syndrome and other metabolicdisorders.

In another aspect the invention provides the pharmaceutical/dietarysupplement/food ingredient compositions comprising at least onecomponent selected from the extract(s), fraction(s), active compound(s)or phytochemical(s) or mixtures thereof derived from Sphaeranthusindicus and at least one component selected from the extract(s),fraction(s), active compound(s) or phytochemical(s) or mixtures thereofderived from Garcinia mangostana, optionally containing one or moreselected from biologically active components derived from plants,animals and microorganisms, pharmaceutically or dietetically acceptableactive ingredients, vitamins, minerals, vehicles, carriers and diluentsor mixtures thereof for the amelioration of the expression or productionof atleast one biomarker protein selected from but not limited to MatrixMetalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3),C-reactive protein (CRP), PPAR-γ, Adipose Differentiation RelatedProtein (ADRP), adipocyte CD36, macrophage CD36, Monocyte Chemotacticprotein (MCP-1), Oxidized LDL, Adipocyte Fatty-acid-Binding Protein(aP2/FABP4/A-FABP), Beta-3 adrenergic receptor (β3-AR), adiponectin,Perilipin and Protein tyrosine phosphatase 1B (PTP 1B).

In another aspect, the invention provides the pharmaceutical/dietarysupplement/food ingredient(s) comprising atleast one component selectedfrom the extract(s) or fraction(s) or active compound(s) orphytochemical(s) or mixtures thereof derived from Sphaeranthus indicus,and the compositions comprising atleast one said component derived fromSphaeranthus indicus in combination with one or more selected frombiologically active components derived from plants, animals andmicroorganisms, pharmaceutically or dietetically acceptable activeingredients, vitamins, minerals, vehicles, carriers and diluents ormixtures thereof for the amelioration of the expression or production ofatleast one biomarker protein related to or associated with metabolicsyndrome, obesity and other metabolic disorders selected from but notlimited to Matrix Metalloproteinase-1 (MMP-1), MatrixMetalloproteinase-3 (MMP-3), C-reactive protein (CRP), PPAR-γ, AdiposeDifferentiation Related Protein (ADRP), adipocyte CD36, macrophage CD36,Monocyte Chemotactic protein (MCP-1), Oxidized LDL, AdipocyteFatty-acid-Binding Protein (aP2/FABP4/A-FABP), Beta-3 adrenergicreceptor (β3-AR), adiponectin, Perilipin and Protein tyrosinephosphatase 1B (PTP 1B).

In another aspect, the invention provides, the pharmaceutical/dietarysupplement/food ingredient(s) comprising atleast one component selectedfrom the extract(s) or fraction(s) or active compound(s) orphytochemical(s) or mixtures thereof derived from Sphaeranthus indicus,and their compositions in combination with one or more selected frombiologically active components derived from plants, animals andmicroorganisms, pharmaceutically or dietetically acceptable activeingredients, vitamins, minerals, vehicles, carriers and diluents ormixtures thereof for controlling one or more of the metabolic processesselected from acceleration of lipolysis, inhibition of adipogenesis,inhibition of alpha-amylase and inhibition of alpha-glucosidase.

In another aspect, the invention provides pharmaceutical/dietarysupplement/food ingredient composition comprising at least one componentselected from the extract(s), fraction(s), active compound(s) andphytochemical(s) or mixtures thereof derived from Sphaeranthus indicusin combination with at least one component selected from the extract(s),fraction(s), active compound(s) and phytochemical(s) or mixtures thereofderived from Garcinia mangostana.

In another aspect, the invention provides pharmaceutical/dietarysupplement/food ingredient compositions comprising at least onecomponent selected from the extract(s), fraction(s), active compound(s)and phytochemical(s) or mixtures thereof derived from Sphaeranthusindicus and at least one component selected from the extract(s),fraction(s), active compound(s) and phytochemical(s) or mixtures thereofderived from Garcinia mangostana, optionally containing one or moreselected from biologically active components derived from plants,animals and microorganisms, pharmaceutically or dietetically acceptableactive ingredients, vitamins, minerals, vehicles, carriers and diluentsor mixtures thereof, wherein the percentage of Sphaeranthus indicusderived component in the composition varies in the range from 0.01% to99.9% and the percentage of Garcinia mangostana derived component variesin the range from 99.9% to 0.010%.

In another aspect the invention provides the pharmaceutical/dietarysupplement/food ingredient compositions comprising atleast one componentselected from the extract(s), fraction(s), active compound(s),phytochemicals or mixtures thereof derived from Sphaeranthus indicus incombination with one or more selected from biologically activecomponents derived from plants, animals and microorganisms,pharmaceutically or dietetically acceptable active ingredients,vitamins, minerals, vehicles, carriers and diluents or mixtures thereof,wherein the percentage of Sphaeranthus indicus derived component in thecomposition varies in the range from 0.01% to 99.9%.

In another aspect, the pharmaceutical/dietary supplement/food ingredientcompositions comprising at least one component selected from theextract(s), fraction(s), active compound(s) and phytochemical(s) ormixtures thereof derived from Sphaeranthus indicus and at least onecomponent selected from the extract(s), fraction(s), active compound(s)and phytochemical(s) or mixtures thereof derived from Garciniamangostana, optionally containing one or more selected from biologicallyactive components derived from plants, animals and microorganisms,pharmaceutically or dietetically acceptable active ingredients,vitamins, minerals, vehicles, carriers and diluents or mixtures thereof,comprise atleast one Sphaeranthus indicus derivedfrullanolide/eudesmanoid sesquiterpene compound selected from but notlimited to frullanolides, 7-hydroxyfrullanolide;11α,13-dihydro-3α,7α-dihydroxy-4,5-epoxy-6β,7-eudesmanolide;11α,13-dihydro-7α-acetoxy-3β-hydroxy-6β,7-eudesm-4-enolide;3-keto-β-eudesmol;11α,13-dihydro-3α,7α-dihydroxyeudesm-4-en-6α,12-olide;11α,13-dihydro-3α,7α-dihydroxyfrullanolide;11α,13-dihydro-7α,13-dihydroxyfrullanolide;11α,13-dihydro-7α-hydroxy-13-methaoxyfrullanolide;2α,7α-dihydroxy-4-en-11,13-dihydroeudesm-6,12-olide; 2α-hydroxycosticacid; 3-keto-7α-hydroxyeudesm-4-en-6,12-olide (cryptomeridiol);4-epicryptomeridiol; sphaeranthanolide; 2α-hydroxysphaerantholide;2α-acetoxysphaerantholide; 2α,7α-dihydroxysphaerantholide;2α-acetoxy-7α-hydroxysphaerantholide;2α-acetoxy-5α-hydroxyisosphaerantholide or mixtures thereof, preferably7-α-hydroxy-4,11(13)-eudesmadien-12,6-olide also known as7-hydroxyfrullanolide or related compounds or its analogs derived fromSphaeranthus indicus.

In another aspect, pharmaceutical/dietary supplement/food ingredient(s)comprising atleast one component selected from extract(s), fraction(s),active compound(s) and phytochemical(s) or mixtures thereof derived fromSphaeranthus indicus and their compositions comprising atleast one saidphytochemical component derived from Sphaeranthus indicus in combinationwith one or more selected from biologically active components derivedfrom plants, animals and microorganisms, pharmaceutically ordietetically acceptable active ingredients, vitamins, minerals,vehicles, carriers and diluents or mixtures thereof, comprise atleastone Sphaeranthus indicus derived frullanolide/eudesmanoid sesquiterpenecompound selected from but not limited to frullanolides,7-hydroxyfrullanolide;11α,13-dihydro-3α,7α-dihydroxy-4,5-epoxy-6β,7-eudesmanolide;11α,13-dihydro-7α-acetoxy-3β-hydroxy-6β,7-eudesm-4-enolide;3-keto-β-eudesmol;11α,13-dihydro-3α,7α-dihydroxyeudesm-4-en-6α,12-olide;11α,13-dihydro-3α,7α-dihydroxyfrullanolide;11α,13-dihydro-7α,13-dihydroxyfrullanolide;11α,13-dihydro-7α-hydroxy-13-methaoxyfrullanolide;2α,7α-dihydroxy-4-en-11,13-dihydroeudesm-6,12-olide; 2α-hydroxycosticacid; 3-keto-7α-hydroxyeudesm-4-en-6,12-olide(cryptomeridiol);4-epicryptomeridiol; sphaeranthanolide; 2α-hydroxysphaerantholide;2α-acetoxysphaerantholide; 2α,7α-dihydroxysphaerantholide;2α-acetoxy-7α-hydroxysphaerantholide;2α-acetoxy-5α-hydroxyisosphaerantholide or mixtures thereof, preferably7-α-hydroxy-4,11(13)-eudesmadien-12,6-olide also known as7-hydroxyfrullanolide or related compounds or its analogs derived fromSphaeranthus indicus.

In another aspect, the invention provides the component(s) selected fromthe extract(s) or fraction(s) or active compound(s) or phytochemical(s)or mixtures thereof derived from Sphaeranthus indicus, and theircompositions as described above, wherein the active compounds inSphaeranthus indicus derived component responsible for the prevention,treatment and control of obesity, metabolic syndrome and other metabolicdisorders include, but not limited to, frullanolide/eudesmanoidsesquiterpenes, preferably 7-α-Hydroxy-4, 11 (13)-eudesmadien-12,6-olidealso known as 7-hydroxyfrullanolide.

The 7-hydroxyfrullanolide or the related compounds used for theprevention, treatment and control of obesity, metabolic syndrome andother metabolic disorders or for making the composition of the presentinvention can be naturally derived or can be produced through synthesisor semisynthesis.

In another aspect, the invention provides the Sphaeranthus indicusderived component selected from the extracts, fractions, activecompounds and phytochemicals, or mixtures thereof used for theprevention, treatment and control of obesity, metabolic syndrome andother metabolic disorders or for making the compositions described abovecomprises, wherein the concentration of active compound7-hydroxyfrullanolide/other frullanolide/eudesmanoidsesquiterpene(s)/other phytochemicals in the extract(s) and fraction(s)derived from Sphaeranthus indicus varies in the range of 0.001% to 100%,preferably 0.01 to 99%.

In another aspect, the concentration of the concentration of activecompound 7-hydroxyfrullanolide/other frullanolide/eudesmanoidsesquiterpene(s)/other phytochemicals in the compositions comprisingSphaeranthus indicus derived component as described in the previousembodiments varies in the range from 0.001% to 99%, preferably 0.01 to95% by weight.

In another aspect, the invention provides the compositions comprisingcomponents derived from Garcinia species and preferably Garciniamangostana for the control, prevention and treatment of obesity,metabolic syndrome and other metabolic disorders, wherein the activecompounds include but not limited to xanthones preferably α-mangostinand γ-mangostin.

In another aspect, the invention provides the compositions comprisingatleast one component selected from the extracts, fractions, enrichedfractions, compounds derived from Garcinia mangostana as described inthe previous embodiments, wherein the concentration of active compounds,α-mangostin and γ-mangostin, either individually or jointly in Garciniamangostana derived extract(s) or fraction(s) varies from 0.001% to99.9%.

In another aspect, the invention provides the compositions comprisingatleast one component selected from the extracts, fractions, enrichedfractions, compounds derived from Garcinia mangostana as described inthe previous embodiments, wherein the concentration of active compoundsα-mangostin and γ-mangostin in the compositions varies eitherindividually or together from 0.001% to 99%, preferably 0.01 to 95%

In another aspect, the invention provides the pharmaceutical/dietarysupplement/food ingredient compositions comprising at least onecomponent selected from the extract(s), fraction(s), active compound(s)and phytochemical(s) or mixtures thereof derived from Sphaeranthusindicus and at least one component selected from the extract(s),fraction(s), active compound(s) and phytochemical(s) or mixtures thereofderived from Garcinia mangostana, for the amelioration of the expressionor production of atleast one metabolic biomarker molecule selected frombut not limited to Matrix Metalloproteinase-1 (MMP-1), MatrixMetalloproteinase-3 (MMP-3), C-reactive protein (CRP), PPAR-γ, AdiposeDifferentiation Related Protein (ADRP), adipocyte CD36, macrophage CD36,Monocyte Chemotactic protein (MCP-1), Oxidized LDL, AdipocyteFatty-acid-Binding Protein (aP2/FABP4/A-FABP), Beta-3 adrenergicreceptor (β3-AR), adiponectin, Perilipin and Protein tyrosinephosphatase 1B (PTP 1B).

In another aspect, the invention provides pharmaceutical/dietarysupplement/food ingredient(s) selected from the extract(s), fraction(s),active compound(s) and phytochemicals or mixtures thereof derived fromSphaeranthus indicus or their compositions, wherein the amelioration ofmetabolic processes responsible for obtaining claimed health benefitscan be selected from but not limited to lipolysis and adipogenesis, fatbreakdown, fat cell regeneration or by any other mechanism associatedwith or related to thereof.

In other embodiment, the invention provides frullanolide/eudesmanolideand related compounds, preferably 7-hydroxyfrullanolide or its analogsor the compositions comprising the said compounds in combination withatleast one component selected from biologically active componentsderived from plants, animals and microorganisms, pharmaceutically ordietetically acceptable active ingredients, vitamins, minerals,vehicles, carriers and diluents or mixtures thereof for the control,prevention and treatment of one or more disease conditions selected frombut not limited to obesity, weight loss, diabetes, atherosclerosis,metabolic syndrome and other metabolic disorders.

In other embodiment, the invention provides, frullanolide/eudesmanolideand related compounds, preferably 7-hydroxyfrullanolide or its analogsor the compositions comprising the said compounds as described above forthe amelioration of the expression or production of atleast onemetabolic biomarker molecules including but not limited to MatrixMetalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3),C-reactive protein (CRP), PPAR-γ, Adipose Differentiation RelatedProtein (ADRP), adipocyte CD36, macrophage CD36, Monocyte Chemotacticprotein (MCP-1), Oxidized LDL, Adipocyte Fatty-acid-Binding Protein(aP2/FABP4/A-FABP), Beta-3 adrenergic receptor (β3-AR), adiponectin,Perilipin and Protein tyrosine phosphatase 1B (PTP 1B)

In another aspect, the invention provides compositions comprisingGarcinia mangostana derived component, wherein said extract(s),fraction(s), active compound(s) and phytochemicals or mixtures thereofderived from Garcinia mangostana exhibit inhibitory effects againstcarbohydrate absorption inhibition enzymes including but not limited toalpha-amylase and alpha-glucosidase and for the inhibition ofadipogenesis.

In another aspect of the invention, the biologically active componentsused for making the compositions described in the previous embodimentscan be selected from the extracts or fractions or pure compounds orphytochemical(s) or powders derived from plants, animals andmicroorganisms having any health benefit selected from but not limitedto anti-diabetic activity, anti-hyperglycemic activity, hypolipidemicactivity, anti-obesity activity, anti-hypertensive activity,anti-platelet aggregation activity, anti-infective activity,anti-atherosclerotic activity and anti-inflammatory activity,anti-oxidant(s) and bio-enhancing activity.

In another aspect, the invention provides pharmaceutical/dietarysupplement/food ingredients derived from Sphaeranthus indicus describedin preceding embodiments, wherein said extract(s) or active fraction(s)or active compound(s) or phytochemicals or mixtures thereof are derivedfrom atleast one of the plant parts selected from but not limited toleaves, flower heads, stem, bark, root, whole plant or mixtures thereof,preferably flower heads.

In another aspect of the invention, pharmaceutical/dietarysupplement/food ingredients and their compositions as described inprevious embodiments, wherein said extract(s) or active fraction(s) oractive compound(s) or phytochemicals or mixtures thereof derived fromSphaeranthus indicus and Garcinia mangostana are obtained throughextraction using solvents selected from one or more of organic solvents,alcohols, hydroalcohols, water or mixtures thereof.

In another aspect of the invention, the extract(s) or active fraction(s)or active compound(s) or phytochemicals or mixtures thereof derived fromGarcinia mangostana are derived from the whole fruit or fruit rind orfruit pulp of Garcinia and preferably Garcinia mangostana.

In another embodiment, the extract(s), fraction(s), active compound(s)and phytochemical(s) or mixtures thereof can be derived from anySphaeranthus species selected from but not limited to Sphaeranthusindicus, Sphaeranthus amaranthoides, S. africanus, S. volgensis, S.kotchyi, S. suaveolens can also used for intended health application orfor preparing components or compositions claimed in the presentinvention.

In another embodiment extract(s), fraction(s), active compound(s) andphytochemical(s) or mixtures thereof used for making the composition canbe derived from other Garcinia species including but not limited toGarcinia cambogia, G. hanburyii, G. schomburgkiana, G. dulcis, G.thorelii, G. xanthochymus, G. cowa, G. bracteata, G. pyrifera and G.nervosa.

The examples of the biologically or pharmaceutically acceptable carriersemployed in the present invention include, but are not limited to,surfactants, excipients, binders, diluents, disintegrators, lubricants,preservatives, stabilizers, buffers, suspensions and drug deliverysystems.

In another aspect, the invention provides pharmaceutical/dietarysupplement/food ingredient compositions, as described in previousembodiments, comprising atleast one component extract(s) or fraction(s)or active compound(s) or phytochemical(s) or mixtures thereof derivedfrom Sphaeranthus indicus in combination with atleast one componentselected from the excipients, carriers and diluents, wherein preferredexamples of solid carriers or diluents or excipients include but notlimited to glucose, fructose, sucrose, maltose, yellow dextrin, whitedextrin, aerosol, microcrystalline cellulose, calcium stearate,magnesium stearate, sorbitol, stevioside, corn syrup, lactose, citricacid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbicacid, dl-alpha-tocopherol, glycerin, propylene glycol, glycerin fattyester, poly glycerin fatty ester, sucrose fatty ester, sorbitan fattyester, propylene glycol fatty ester, acacia, carrageenan, casein,gelatin, pectin, agar, vitamin B group, nicotinamide, calciumpantothenate, amino acids, calcium salts, pigments, flavors andpreservatives and preferred examples of liquid carriers or diluents orexcipients include but not limited to distilled water, saline, aqueousglucose solution, alcohol (e.g. ethanol), propylene glycol andpolyethylene glycol; and oily carriers such as various animal andvegetable oils, white soft paraffin, paraffin and wax.

In another aspect, the invention provides pharmaceutical/dietarysupplement/food ingredient(s) or their composition(s) as claimed inpreceding embodiments, wherein said component or composition isadministered orally, topically or parenterally or by inhalation to asubject or mammal or warm blooded animal in need thereof.

In another aspect, the invention provides pharmaceutical/dietarysupplement/food ingredient(s) or their composition(s) as claimed inpreceding embodiments, wherein said components or compositions can beformulated as oral agents such as tablets, soft capsule, hard capsule,pills, granules, powders, emulsions, suspensions, syrups, pellets, food,beverages and the like; and parenteral agents such as injectionsolution, drops, suppositories and the like; and transdermal agents suchas patches, topical creams and gel, and food ingredients or beverages.

In another aspect, the invention provides a method for thecontrol/prevention/treating of a disease condition selected from but notlimited to obesity, Metabolic Syndrome and other metabolic disorderscomprising administering to a subject in need thereof a therapeuticallyeffective amount of atleast one pharmaceutical/dietary supplement/foodingredient selected from the extract(s), fraction(s), activecompound(s), phytochemical(s) or mixtures thereof derived fromSphaeranthus indicus or their composition(s) as described in preceedingembodiments.

In another aspect, the invention provides provides a method of promotinglipolysis and/or inhibiting adipogenesis comprising administering to asubject or mammal or warm blooded animal in need thereof atherapeutically effective quantity of atleast one pharmaceutical/dietarysupplement/food ingredient(s) derived from Sphaeranthus indicus or theircomposition(s) as described in preceeding embodiments.

A method of inhibiting adipogenesis or inhibiting digestive enzymesselected from but not limited to alpha-amylase and/or alpha-glucosidasecomprising administering to a subject or mammal or warm blooded animalin need thereof a therapeutically effective quantity of atleast onecomponent selected from the extract(s), fraction(s), active compound(s),phytochemical(s) or mixtures thereof derived from Garcinia mangostana orthe compositions comprising the said G. mangostana derived component(s).

In another aspect, the invention provides a method of treating obesity,diabetes, metabolic syndrome or other metabolic disorders comprisingadministering to a subject or animal in need thereof a therapeuticallyeffective amount of atleast one component selected from extract(s) orfraction(s) or active compound(s) or phytochemical(s) or mixturesthereof derived from Sphaeranthus indicus or their compositions,preferably in combination with at least one component selected from theextract(s), fraction(s) and active compound(s) or mixtures thereofderived from Garcinia mangostana, optionally containing one or more ofbiologically active components derived from plants, animals andmicroorganisms, pharmaceutically or dietetically acceptable activeingredients, vitamins, minerals, vehicles, carriers and diluents ormixtures thereof.

In another aspect, the invention provides a method of using componentsselected from the extract(s) or fraction(s) or active compound(s) orphytochemical(s) or mixtures thereof derived from Sphaeranthus indicusand their compositions for the amelioration the expression or productionof biological markers selected from but not limited to MatrixMetalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3),C-reactive protein (CRP), PPAR-γ, Adipose Differentiation RelatedProtein (ADRP), adipocyte CD36, macrophage CD36, Monocyte Chemotacticprotein (MCP-1), Oxidized LDL, Adipocyte Fatty-acid-Binding Protein(aP2/FABP4/A-FABP), Beta-3 adrenergic receptor (β3-AR), adiponectin,Perilipin and Protein tyrosine phosphatase 1B (PTP 1B).

In another aspect, the invention provides a method of amelioration theexpression of biological markers selected from but not limited to MatrixMetalloproteinase-1 (MMP-1), Matrix Metalloproteinase-3 (MMP-3), PPAR-γ,Adipose Differentiation Related Protein (ADRP), adipocyte CD36,macrophage CD36, Monocyte Chemotactic protein (MCP-1), Oxidized LDL,Adipocyte Fatty-acid-Binding Protein (aP2/FABP4/A-FABP), Beta-3adrenergic receptor (β3-AR), adiponectin, Perilipin and Protein tyrosinephosphatase 1B (PTP 1B) comprising administering to a subject or ananimal in need thereof a therapeutically effective amount of atleast onecomponent selected from the extract(s) or fraction(s) or activecompound(s) or phytochemical(s) or mixtures thereof derived fromSphaeranthus indicus and their compositions as described in thepreceeding embodiments.

In another aspect, the invention provides phytochemical components suchas extract(s) or fraction(s) or active compound(s) or phytochemical(s)or mixtures thereof derived from Sphaeranthus indicus or thecompositions comprising the said Sphaeranthus indicus derived componentsfor ameliorating one or more metabolic processes selected from promotinglipolysis, inhibiting adipogenic activity, fat breakdown, fat cellregeneration or by any other mechanism associated with or related tothereof.

In another aspect, the invention provides components such as extract(s)or active fraction(s) or active compound(s) or phytochemical(s) ormixtures thereof derived from Garcinia mangostana and their compositionsfor inhibiting carbohydrate break-down enzymes such as alpha-amylaseand/or alpha-glucosidase.

In another aspect, the invention provides components such as extract(s)or fraction(s) or active compound(s) or phytochemical(s) or mixturesthereof derived from Garcinia mangostana for treating obesity andmetabolic syndrome through anti-adipogenic activity.

In another aspect, the invention provides compositions comprising atleast components selected from the extract(s) or fraction(s) or activecompound(s) or phytochemical(s) or mixtures thereof derived fromSphaeranthus indicus and at least one component selected from theextract(s) or active fraction(s) or active compound(s) orphytochemical(s) or mixtures thereof derived from Garcinia mangostana,for digestive enzyme inhibition/carbohydrate absorption inhibition suchas alpha-amylase inhibition, alpha-glucosidase inhibition for theprevention and treatment of obesity, diabetes, metabolic syndrome ordisease conditions associated with metabolic syndrome.

In another aspect, the invention provides the pharmaceutical/dietarysupplement/food ingredient(s) selected from the extract(s), fraction(s),active compound(s) or phytochemical(s) or mixtures thereof derived fromSphaeranthus indicus and their compositions in combination with at leastone component selected from the extract(s), fraction(s), activecompound(s) or phytochemical(s) or mixtures thereof derived fromGarcinia mangostana as described in preceeding embodiments foranti-adipogenic activity, promoting lipolysis, fat breakdown, fat cellregeneration or by any other mechanism associated with or related tothereof.

In a further embodiment of the present invention, the componentsselected from extract(s), fraction(s), active compound(s) orphytochemical(s) or mixtures thereof derived from Sphaeranthus indicusor their compositions as described above can be optionally combined withbio-availability enhancing agents selected from but not limited toextract(s), fraction(s), pure compound(s) derived from Piper nigrumextract(s) and Piper longum, piperine.

In alternative aspect of the invention, the components selected fromextract(s), fraction(s), active compound(s) or phytochemical(s) ormixtures thereof derived from Sphaeranthus indicus or their compositionsclaimed in the present invention are delivered in the form of controlledrelease tablets, using controlled release polymer-based coatings by thetechniques including nanotechnology, microencapsulation, colloidalcarrier systems and other drug delivery systems known in the art. Thesaid formulation can be designed for once a daily administration ormultiple administrations per day.

In other aspect of the invention, the components selected fromextract(s), fraction(s), active compound(s) or phytochemical(s) ormixtures thereof derived from Sphaeranthus indicus or their compositionsclaimed in the present invention can also be formulated into or added toexisting or new food and beverage form(s) and animal feeds as a healthyfood or beverage or feed for control, prevention and treatment ofseveral diseases including but not limited to obesity, weight loss,diabetes, atherosclerosis, arteriosclerosis, cardiovascular diseases,neurological disorders, Alzheimer's, cognitive disorders, oxidativestress, skin disorders, aging of the skin, UV irradiated damage,hypertension, hypercholesteremia (LDL, HDL, VLDL), hyperlipidemia(triglycerides), immune deficiency, cancer, metabolic syndrome and othermetabolic disorders.

The unexpected and superior ameliorating effects of the extract(s),fraction(s) and active compound(s) or phytochemical(s) or mixturesthereof derived from Sphaeranthus indicus and their compositions incombination with extract(s), fraction(s), active compound(s) orphytochemical(s) or mixtures thereof derived from Garcinia mangostana onthe enzymes, metabolic biological markers and metabolic processesrelated to one or more of obesity, metabolic syndrome and othermetabolic disorders are illustrated by the following non-limitingexamples:

Example 1 Sphaeranthus indicus Ethyl Acetate Extract (LI/DD-II/054A/01)

Sphaeranthus indicus flower heads (2.2 kg) were charged into a pilotextractor and extracted with ethyl acetate (22 L) at reflux temperaturefor 2 h. The extract was filtered and the spent raw material wasre-extracted twice with ethyl acetate (2×13 L) under similar conditions.The combined extract was fine filtered and concentrated over a climbingfilm evaporator to obtain residue (174 g). The ethyl acetate extractshowed 11% of 7-hydroxy-4, 11 (13)-eudesmadien-12,6-olide(7-hydroxyfrullanolide) by HPLC method of analysis.

Example 2 Sphaeranthus indicus Hexane Extract (LI/DD-II/054A/02)

Sphaeranthus indicus flower heads (1.0 kg) were taken in a Soxhletapparatus and extracted with hexane (6 L) at reflux temperature for 4 h.The extract was fine filtered and the spent raw material wasre-extracted twice with hexane (2×4 L). The extracts were combined andconcentrated under vacuum to obtain a residue (43 g). The hexane extractshowed 21% of 7-hydroxyfrullanolide by HPLC method of analysis.

Example 3 Sphaeranthus indicus Methanol Extract (LI/DD-II/054A/03)

Sphaeranthus indicus flower heads (1 kg) were taken in a RB flask andextracted with methanol (8 L) at 80° C. temperature for 2 h. The extractwas filtered and the spent raw material was re-extracted twice withethyl acetate (2×6 L) under similar conditions. The combined extract wasfine filtered and concentrated over a climbing film evaporator to obtaina residue (110 g). The methanol extract (LI/DD-II/054A/03) showed 8.3%of 7-hydroxyfrullanolide by HPLC method of analysis.

Example 4 Purification of 7-Hydroxyfrullanolide (LI054A01)

The ethyl acetate extract (90 g) of the flower heads of Sphaeranthusindicus was subjected to chromatography over a silica column usingeluants of increasing polarity from hexane to acetone. The fractionseluted with 20% acetone/hexane were combined and evaporated under vacuumto give a residue (16 g) containing 63% of 7-hydroxyfrullanolide. Theresidue was subjected to re-chromatography over silica using solvents ofincreasing polarity from hexane to ethyl acetate. The fractions elutedwith 20-25% ethyl acetate/hexane yielded were combined and evaporatedand the residue was precipitated from acetonitrile to obtain semi-pure7-hydroxyfrullanolide (91%). The residue was finally purified on silicacolumn using chloroform/hexane mixtures. The fractions eluted with25-35% chloroform/hexane were combined and evaporated to give pure7-hydroxyfrullanolide (7.3 g, 99%).

Example 5 Garcinia mangostana Methanol Extract (AR 933)

Shade dried fruit rind (1 Kg) of Garcinia mangostana was pulverized tocoarse powder, and extracted with methanol (5 L) for 2 hrs at 60-65′C.The solvent was separated from the raw material by filtration.Extraction process was repeated thrice using methanol (2×3 L & 1×2 L).The combined extracts were fine filtered and concentrated under reducedpressure and allowed to precipitate at ambient temperature. The solidseparated was filtered to give a dry powder (165 g, α-Mangostin: 32% andγ-Mangostin: 3%).

Example 6 Purification of α-mangostin and γ-mangostin

The methanol extract (90 g) of G. mangostana having 32% α-mangostin wassubjected to chromatography over a silica column using eluants ofincreasing polarity from chloroform to methanol. The fractions elutedwith 10-15% methanol/chloroform were monitored and the fractioncontaining α-mangostin were combined and evaporated under vacuum and theresidue was crystallized from dichloromethane/methanol mixture to obtaina residue (20 g) containing 99% of α-mangostin. The fractions elutedwith 25% methanol/chloroform were monitored and the fraction containingγ-mangostin were combined and evaporated under vacuum and the residuewas crystallized from dichloromethane/methanol mixture to residue (2.1g) containing 99% of γ-mangostin.

Example 7 Process for Preparing Composition 1A, Composition 1B,Composition 1C and Composition 1D

Composition-1A was prepared by mixing unit doses of the followingcomponents: Three parts of Sphaeranthus indicus ethyl acetate extract(LI/DD-II/054A/01, 3 g) and one part of Garcinia mangostana methanolextract (AR933, 1 g).

Composition 1B was prepared by mixing unit doses of the followingcomponents: One part of Sphaeranthus indicus ethyl acetate extract (1 g)and three parts of Garcinia mangostana methanol extract (3 g).

Composition 1C was prepared by mixing unit doses of the followingcomponents: one part of Sphaeranthus indicus methanol extract (1 g) andthree parts of Garcinia mangostana methanol extract (3 g).

Composition 1D was prepared by mixing unit doses of the followingcomponents: three parts of Sphaeranthus indicus methanol extract (3 g)and one part of Garcinia mangostana methanol extract (1 g)

Example 8 Process for Preparing Compositions 2A, 2B and 2C

Composition 2A: Composition 2A was prepared by mixing unit doses of thefollowing components:

Two parts of Sphaeranthus indicus ethyl acetate extract (2 g) and

One part of Garcinia mangostana methanol extract (1 g)

Composition 2B: Composition 2B was prepared by mixing unit doses of thefollowing components:

One part of Sphaeranthus indicus ethyl acetate extract (1 g) and

Two parts of Garcinia mangostana methanol extract (2 g)

Composition 2C: Composition 2C was prepared by mixing unit doses of thefollowing components:

One part of Sphaeranthus indicus ethyl acetate extract (1 g) and

One part of Garcinia mangostana methanol extract (1 g)

Example 9 Process for Preparing Compositions 2D and 2E

Composition 2D: Composition 2D was prepared by mixing unit doses of thefollowing components:

Four parts of Sphaeranthus indicus ethyl acetate extract (4 g) and

One part of Garcinia mangostana methanol extract (1 g).

Composition 2E: Composition 2E was prepared by mixing unit doses of thefollowing components:

One part of Sphaeranthus indicus ethyl acetate extract (1 g) and

Four parts of Garcinia mangostana methanol extract (4 g).

Example 10 Inhibition of Matrix Metalloproteinase-1 (MMP-1) Productionby Gorakhmundi Extract

MMP-1 was evaluated in PMA induced human melanoma cells, A2058. Briefly,the cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) with2 mM Glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10%fetal bovine serum (Hyclone, Logan, Utah). Five thousand cells per wellwere seeded into a 96-well cell culture plate (Corning, USA) one daybefore the experiment. The culture media was replaced with fresh DMEMcontaining 10% fetal bovine serum. Sphaeranthus indicus ethyl acetateextract (LI/DD-II/054A/01) was serially diluted in medium, ranging from0.1 to 10 μg/ml and was pre-incubated with cells for 2 hour at 5% CO2 at37° C., and then stimulated with 50 mM of PMA for 24 hours. Thesupernatant was harvested and used to measure MMP-1 production by MMP-1ELISA Development Kit (R&D System, Minneapolis, Minn., USA). The MMP-1concentration in culture supernatant was estimated quantitatively byinterpolating the optical densities into the standard curve generatedfrom known concentrations of MMP-1.

The inhibitory effect at different concentration of ethyl acetateextract (LI/DD-II/054A/01) of Sphaeranthus indicus is depicted inFIG. 1. The inhibitory concentration for 50% inhibition (IC₅₀) of MMP-1was calculated from the plot constructed by plotting percentageinhibition against concentration. The ethyl acetate extract(LI/DD-II/054A/01) of Sphaeranthus indicus showed an IC₅₀ value of 10.14μg/mL. Using a similar procedure the IC₅₀ value of the pure compound7-hydroxyfrullanolide (LI054A01) was found to be 1.2 μg/mL.

Example 11 Inhibition of Matrix Metalloproteinase-3 (MMP-3) Productionby Gorakhmundi Extract

MMP-3 was evaluated in Interleukin-1β induced human lung tumor cell lineA549. Briefly, the cells were cultured in DMEM with 2 mM Glutamine, 100U/mL penicillin, 100 mg/mL streptomycin and 10% fetal bovine serum(Hyclone, Logan, Utah). Five thousand cells per well were seeded into a96-well cell culture plate (Corning, USA) one day before the experiment.The culture media was replaced with fresh DMEM containing 10% fetalbovine serum. Sphaeranthus indicus ethyl acetate extract(LI/DD-II/054A/01) was serially diluted in medium, ranging from 0.1 to10 μg/ml and was pre-incubated with cells for 2 hour at 5% CO₂ at 37°C., and then stimulated with 10 ng/mL human IL-1β (R&D System,Minneapolis, Minn.) for 24 hours. The supernatant was harvested and usedto measure MMP-3 production by ELISA development kit (R&D System,Minneapolis, Minn., USA). The MMP-3 concentration in culture supernatantwas estimated quantitatively by interpolating the optical densities intothe standard curve generated from known concentrations of MMP-3. Theinhibitory concentration for 50% inhibition (IC₅₀) of MMP-3 wascalculated from the plot constructed by plotting percentage inhibitionagainst concentration. The ethyl acetate extract (LI/DD-II/054A/01) ofSphaeranthus indicus showed an IC₅₀ value of 0.36 μg/mL against MMP-3.The data is summarized in FIG. 2. Using a similar procedure the IC₅₀value of the pure compound 7-hydroxyfrullanolide (LI054A01) was found tobe 0.075 μg/mL.

Example 12 Assessment of Inhibition of Lipid Accumulation inDifferentiated Adipocytes by Sphaeranthus indicus Ethyl Acetate Extract(LI/DD-II/054A/01) and 7-Hydroxyfrullanolide (LI054A01)

One hundred thousand 3T3-L1 Human pre-adipocyte cells in Dulbecco'sModified Eagles Medium (DMEM) containing 10% Fetal Bovine Serum (FBS)were taken into each well of a 24-well plate and incubated for 48 h at37° C. and 5% CO₂. The differentiation of pre-adipocyte cells wasinitiated in a differentiation medium containing 10 μg/ml insulin, 1.0μM dexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX) for 48 h.After this the medium was replaced by DMEM containing 10 μg/ml insulinand incubated for 3 days. Then the differentiating cells were treated 10μg/ml of Sphaeranthus indicus ethyl acetate extract (LI/DD-II/054A/01)and 0.5 μg/ml of 7-hydroxyfrullanolide (LI054A01) and maintained in themedium for another 3-5 days. The cells incubated with 0.1% DMSO wereconsidered as the vehicle control. After the incubation period, cellswere washed with phosphate buffered saline (PBS) and fixed with 10%buffered formalin for 1 h at room temperature. One small aliquot of cellsuspension was separated for cell counting in hemocytometer chamber.Fixed cells were stained with Oil Red O solution to measure the cellularneutral lipid accumulation. Briefly, cells were washed with PBS, fixedwith 10% buffered formalin and stained with Oil Red O solution (0.5 g in100 ml isopropanol) for 10 min. After removing the staining solution,the dye retained in the cells will be eluted into isopropanol and ODmeasured at 550 nm. The inhibition of fat accumulation in the treatedcells was compared with the mock treated differentiated adipocytes. Thetreated and control cells were also analyzed and compared for inhibitionof lipid accumulation visually under microscope and recorded digitallyin suitable image capture system. The anti-adipogenic activities shownby Sphaeranthus Indicus ethyl acetate extract (LI/DD-II/054A/01) andLIO054A01 are summarized in the following table.

Anti-Adipogenic Activity of Sphaeranthus indicus

TABLE I % inhibition of S. No Name of the product lipid accumulation 1LI/DD-II/054A/01 65.9% at 10 μg/ml 2 LI054A01 68.7% at 0.5 μg/ml

Example 13 Assessment of Pro-Lipolytic Activity of Sphaeranthus IndicusEthyl Acetate Extract (LI/DD-II/054A/01) and 7-Hydroxyfrullanolide(LI054A01) in Differentiated Adipocytes

The lipolytic activity was assessed in mature adipocytes as per theprocedure of Chemicon International, USA, by measuring free glycerolsecreted into the culture medium. One hundred thousand 3T3-L1 Humanpre-adipocyte cells in Dulbecco's Modified Eagles Medium (DMEM)containing 10% Fetal Bovine Serum (FBS) were taken into each well of a24-well plate and incubated for 48 h at 37° C. and 5% CO₂. Thedifferentiation of pre-adipocyte cells was initiated in adifferentiation medium containing 10 μg/ml insulin, 1.0 μMdexamethasone, and 0.5 mM isobutylmethylxanthine (IBMX). The cells weredifferentiated for 5 days and then the culture medium was removed. Themonolayer was washed twice with wash solution (Hank's balanced saltsolution), and then 250 μL of incubation solution (Hank's balanced saltsolution plus 2% bovine serum albumin) was added to the wells intriplicate in presence or absence of Sphaeranthus indicus ethyl acetateextract (LI/DD-II/054A/01) and 7-hydroxyfrullanolide (LI054A01), and thecells were further incubated for 16 h. To measure lipolysis, 200 μL offree glycerol assay reagent was added to 25 μL of culture supernatantsand controls containing glycerol standard. The samples and the controlswere incubated for 15 min, and the absorbance was read at 540 nm. Astandard curve constructed from the glycerol was used to calculate theconcentration of free glycerol in the culture supernatants. Thepercentage increase in glycerol concentration in the sample solutionscompared to the control containing the known concentrations of glycerolcorresponds to the percentage acceleration of lipolysis byLI/DD-II/054A/01 or LI054A01. The percentage increase in lipolysisaccelerated by LI/DD-II/054A/01 and LI054A01 are summarized table II.

Pro-Lipolytic Activity of Sphaeranthus indicus

TABLE II % acceleration of S. No Name of the product lipolysis 1LI/DD-II/054A/01 26.7% at 25 μg/ml 2 LI054A01 47.8% at 5 μg/ml

Example 14 Inhibition of Peroxisome Proliferator-Activated ReceptorGamma (PPARγ)_, Adipose Differentiation Related Protein (ADRP), CD36,Adipocyte Fatty Acid Binding Protein (aP2), Beta-3 Adrenergic Receptor(β3AR) and Perilipin in 3T3-L1 Adipopcytes by Sphaeranthus indicus EthylAcetate Extract (LI/DD-II/054A/01) and 7-Hydroxyfrullanolide (LI054A01)

Experimental protocol: Mouse pre-adipocyte 3T3-L1 cells are maintainedin Dulbecco's Modified Eagles Medium (DMEM) supplemented with 2 mMglutamine, 4.5 g/L glucose and 10% fetal bovine serum. Equal number ofcells was plated in each well of 24-well culture plates. Cells werepre-treated separately with 2.5, 5 and 10 μg/mL of LI/DD-II/054A/01 or 1μg/mL 7-hydroxyfrullanolide for 2 h and followed by addition ofdifferentiation medium containing 500 nM insulin, 1.0 μM dexamethasone,and 0.5 mM isobutylmethylxanthine (IBMX) for 48 h. Thereafter, cellswere further incubated with post differentiation medium (DMEM containing100 nM insulin) in presence or absence of Sphaeranthus indicus extractLI/DD-II/054A/01 extract and 7-hydroxyfrullanolide (LI054A01). Finally,the cells were harvested, washed with chilled phosphate buffered salineand lysed with the lysis buffer. The protein extracts were clarified at14,000 g for 20 min. Protein content was measured in Bradford method byusing Coomassie blue dye and cell lysates were stored in aliquots at−80° C. until further use. The modulation of adipocyte differentiationmarkers such as Peroxisome proliferator-activated receptor gamma(PPARγ), CD36, adipocyte fatty acid binding protein (aP2); andintracellular lipid droplet surface associated protein, perilipinexpression were evaluated by immunoblot assay.

Inhibition of protein expression of biomarker molecules adipocytes inpresence or absence of Sphaeranthus indicus extract LI/DD-II/054A/01 and7-hydroxyfrullanolide (LI054A01) was evaluated in immunoblot assay.Briefly, equal amount of cell lysates proteins were resolved in 7.5%SDS-PAGE; thereafter, the proteins were transferred to nitrocellulosemembrane. After blocking the non-specific sites, the membrane wasincubated with either anti-PPARγ, or anti-CD36, or anti-aP2, oranti-β3AR, or anti-ADRP, or anti-perilipin antibody. Finally, thespecific immuno-reactive bands were developed with West-picochemiluminescent substrate (Pierce Biotechnology, IL, USA), and theimmunoblot images were recorded in a Kodak Image Station (Kodak, USA).Band intensities were calculated densitometrically and normalized withexpression of actin in respective samples. The data is summarized inFIG. 3

Example 15 Inhibition of CD36 Production by Sphaeranthus indicus ExtractEthyl Acetate Extract (LI/DD-II/054A/01) and 7-Hydroxyfrullanolide(LI054A01) in Macrophage Cells

Experimental protocol: This was evaluated in glucose induced J774, mousemacrophage cells. Briefly, the cells were cultured in DMEM with 2 mMGlutamine, 100 U/mL penicillin, 100 mg/mL streptomycin and 10% fetalbovine serum (Hyclone, Logan, Utah). Equal number of cells was seededinto 35 mm petri dishes (Corning, USA) one day before the experiment.The culture media was replaced with fresh, glucose free DMEMsupplemented with 10% fetal bovine serum. Sphaeranthus indicus ethylacetate extract (LI/DD-II/054A/01) was serially diluted in above culturemedium, ranging from 1 to 10 μg/ml and LI054A01 was diluted at 1 g andall cultures were pre-incubated for 2 hours at 5% CO₂ at 37° C., andthen incubated with 600 mg/dL of glucose for 5 days. The control culturewas supplemented with 100 mg/dL glucose. The cells were harvested andlysed with lysis buffer. Cell lysates were clarified at 14,000 g.Protein concentration was measured by Bradford method.

Inhibition of CD36 protein expression in high glucose induced J774macrophage cells in presence or absence of LI/DD-II/054A/01 and LI054A01was evaluated in immunoblot assay. Briefly, equal amount of cell lysatesproteins were resolved in 7.5% SDS-PAGE; thereafter, the proteins weretransferred to nitrocellulose membrane. After blocking the non-specificsites, the membrane was incubated with CD36 antibody (R&D Systems,Minneapolis, Minn.). Finally, the specific immuno-reactive bands weredeveloped with West-pico chemiluminescent substrate (PierceBiotechnology, IL, USA), and the immunoblot images were recorded in aKodak Image Station (Kodak, USA). Band intensities were calculateddensitometrically and normalized with expression of actin in respectivesamples. The results are summarized in FIG. 4.

Example 16 Assessment of Inhibition of Lipid Accumulation inDifferentiated Adipocytes by Garcinia mangostana Methanol Extract (AR933), α-Mangostin and γ-Mangostin

The anti-adipogenic activity of Garcinia mangostana extract (AR 933),α-mangostin and γ-mangostin were assessed by method as described inexample 12 above. The anti-adipogenic activities shown by the extract AR933, α-mangostin and γ-mangostin are summarized in the following table.

Anti-Adipogenic Activity of Garcinia Mangostana

TABLE III % inhibition of Lipid accumulation S. No Name of the product10 μg/ml 1 α-mangostin 16.06 2 γ-mangostin 61.57 3 G. mangostana (AR933) 48.5

Example 17 Assessment of Pro-Lipolytic α-Mangostin, γ-Mangostin andGarcinia Mangostana Methanol Extract (AR 933) in DifferentiatedAdipocytes

The lipolytic activity was assessed in mature adipocytes as described inthe Example 13 above. The percentage increase in glycerol concentrationin the sample solutions compared to the control containing the knownconcentrations of glycerol corresponds to the percentage acceleration oflipolysis for the test compounds. The percentage increase in lipolysisaccelerated by α-mangostin, γ-mangostin and Garcinia mangostana methanolextract (AR 933) are summarized in the following table.

Pro-Lipolytic Activity of Garcinia Mangostana

TABLE IV % acceleration S. of lipolysis No Name of the product 25 μg/ml1 α-mangostin 156.56 2 γ-mangostin 4.52 3 G. mangostana (AR 933) 55.8

Example 18 Inhibition of Alpha-Amylase by Garcinia mangostin MethanolExtract (AR 933), α- and γ-Mangostins

α-Amylase inhibitory activity was measured using the dinitrosalicylicacid (DNS) method developed by Bernfeld (Methods in Enzymology, 1955,Vol. 1, pp 149-158), improved by Jamieson et al (Journal of DentalResearch 1969; 48(3): 483) and adopted for testing inhibitory potentialof test substances by M. C. M da Silva et al (2004, Pesq. Agropec.bras., Brasilia, 2004; 39(3); pp 201-208) using 1% soluble starch assubstrate.

The test substances (α-mangostin, γ-mangostin and AR 933) werepre-incubated with α-amylase 100 μL (10-25 U/mL) at room temperature for20 minutes prior to the addition of 100 μL of the substrate solutionfollowed by incubation at 37° C. for 10 minutes. The reactions werestopped by the addition of 200 μL of DNS reagent, followed by colordevelopment by placing the tubes in boiling water for 5 minutes. Afteraddition of 3.6 mL distilled water, the absorbance was read at 470 nm.Known α-amylase inhibitor was used as positive control and vehicle wasused as negative control. Assays were carried out at least in duplicate.Percentage inhibition will be calculated by comparing mean test OD withmean control OD. {% inhibition=[(COD−TOD)/COD]×100}. The IC₅₀ valueswere calculated by linear regression analysis of the dose responsecurve. The results are summarized in the following table.

TABLE V IC₅₀ (μg/ml) for Compound α-amylase γ-mangostin 3.88 α-mangostin3.99 AR 933 5.32 Acarbose 12.7

Example 19 Inhibition of α-Glucosidase by Garcinia Mangostin MethanolExtract (AR 933), α and γ Mangostins

α-Glucosidase inhibitory activity was measured using the in vitro methoddeveloped by D. Prasanth et al., (Fitoterapia 2001, 72, 686-688). In amicro plate well was taken 50 μL of α-glucosidase enzyme (0.4 U/mL), andtreated with 90 μL of 100 mM phosphate buffer (pH 7) and 10 μL testsubstances (α-mangostin, γ-mangostin and AR 933) or vehicle control. Thecontents were mixed well and the reaction mixtures were incubated atroom temperature for 5 min, and then added 50 μL of p-nitrophenylα-D-glucopyranose (20 mM) as substrate. The contents were mixed well andagain incubated at room temperature for 15 min. The reaction was stoppedby the addition of 30 μL of sodium carbonate solution (135 mM). Theabsorbance was measured at 405 nm using micro plate reader. Control andtest blank ODs were obtained by replacing enzyme with buffer. Percentageinhibitions were calculated by comparing mean test OD with mean controlOD. {% inhibition=[(COD−TOD)/COD]×100}. The IC₅₀ values were calculatedby linear regression analysis of the dose response curve.

TABLE VI IC₅₀ (μg/ml) for Compound α-glucosidase γ-mangostin 0.16α-mangostin 0.30 AR 933 0.29 Green Tea 1.10

Example 20 Inhibition of PPARγ_, ADRP, CD36, aP2, β3AR and Perilipin in3T3-L1 Adipocytes by Garcinia mangostana Extract (AR 933)

Experimental protocol: Mouse pre-adipocyte 3T3-L1 cells are maintainedin Dulbecco's Modified Eagles Medium (DMEM) supplemented with 2 mMglutamine, 4.5 g/L glucose and 109/o fetal bovine serum. Equal number ofcells was plated in each well of 24-well culture plates. Cells werepre-treated separately with 2.5 and 5 μg/mL of Garcinia mangostanamethanol extract (AR 933) for 2 h and followed by addition ofdifferentiation medium containing 500 nM insulin, 1.0 μM dexamethasone,and 0.5 mM isobutylmethylxanthine (IBMX) for 48 h. Thereafter, cellswere further incubated with post differentiation medium (DMEM containing100 nM insulin) in presence or absence of Garcinia mangostana methanolextract (AR 933). Finally, the cells were harvested, washed with chilledphosphate buffered saline and lysed with the lysis buffer. The proteinextracts were clarified at 14,000 g for 20 min. Protein content wasmeasured in Bradford method by using Coomassie blue dye and cell lysateswere stored in aliquots at −80° C. until further use. The modulation ofadipocyte differentiation markers such as PPARγ, CD36, aP2 andintracellular lipid droplet surface associated protein, perilipinexpression were evaluated by immunoblot assay.

Inhibition of protein expression of biomarker molecules adipocytes inpresence or absence of Garcinia mangostana extract was evaluated inimmunoblot assay. Briefly, equal amount of cell lysates proteins wereresolved in 7.5% SDS-PAGE; thereafter, the proteins were transferred tonitrocellulose membrane. After blocking the non-specific sites, themembrane was incubated with either anti-PPARγ, or anti-CD36, oranti-aP2, or anti-β3AR, or anti-ADRP, or anti-perilipin antibody.Finally, the specific immuno-reactive bands were developed withWest-pico chemiluminescent substrate (Pierce Biotechnology, IL, USA),and the immunoblot images were recorded in a Kodak Image Station (Kodak,USA). Band intensities were calculated densitometrically and normalizedwith expression of actin in respective samples. The data is summarizedin FIG. 5.

Example 21 Assessment of Inhibition of Lipid Accumulation inDifferentiated Adipocytes by Compositions 1B, 2C and 2E ComprisingLI/DD-II/054A/01 and AR 933

The anti-adipogenic activities of Compositions 1B, 2C and 2E were alsoassessed by methods as described in example 12 above. Theanti-adipogenic activities shown by Compositions 1B, 2C and 2E aresummarized in Table VII.

Anti-Adipogenic Activity of Compositions 1B, 2C & 2E

TABLE VII % Inhibition of Lipid accumulation S. No Name of the product10 μg/mL 1 Composition 1B (1:3) 23.6 2 Composition 2C (1:1) 21.5 3Composition 2E (1:4) 38.6

Example 22 Assessment of Pro-Lipolytic Compositions 1B, 2C and 2EComprising LI/DD-II/054A/01 and AR 933 in Differentiated Adipocytes

The lipolytic activity of Compositions 1B, 2C & 2E were assessed inmature adipocytes as described in example 13 above. The percentageincrease in glycerol concentration in the sample solutions compared tothe control containing the known concentrations of glycerol correspondsto the percentage acceleration of lipolysis by Compositions 1B, 2C & 2E.The percentage increase in lipolysis accelerated by Compositions 1B, 2Cand 2E are summarized in the following table

Pro-Lipolytic Activity of Compositions 1B, 2C and 2E

TABLE VIII % acceleration % acceleration of lipolysis of lipolysis S. NoName of the product 25 μg/ml 50 μg/ml 1 Composition 1B (1:3) 31.7 59.8 2Composition 2C (1:1) 21.9 46.9 3 Composition 2E (1:4) 37.95 71.4

Example 23 Inhibition of PPARγ_, ADRP, CD36, aP2, β3AR and Perilipin in3T3-L1 Adipocytes by Composition 1B

Experimental protocol: Mouse pre-adipocyte 3T3-L1 cells are maintainedin Dulbecco's Modified Eagles Medium (DMEM) supplemented with 2 mMglutamine, 4.5 g/L glucose and 10% fetal bovine serum. Equal number ofcells was plated in each well of 24-well culture plates. Cells werepre-treated with 5 μg/ml of either LI/DD-II/054A/01 or AR 933 orcomposition 1B for 2 h and followed by addition of differentiationmedium containing 500 nM insulin, 1.0 μM dexamethasone, and 0.5 mMisobutylmethylxanthine (IBMX) for 48 h. Thereafter, cells were furtherincubated with post differentiation medium (DMEM containing 100 nMinsulin) in presence or absence of composition 1B. Finally, the cellswere harvested, washed with chilled phosphate buffered saline and lysedwith the lysis buffer. The protein extracts were clarified at 14,000 gfor 20 min. Protein content was measured in Bradford method by usingCoomassie blue dye and cell lysates were stored in aliquots at −80° C.until further use. The modulation of adipocyte differentiation markerssuch as Peroxisome proliferator activator receptor-gamma (PPAR-γ), CD36,adipocyte fatty acid binding protein (aP2); and intracellular lipiddroplet surface associated protein, perilipin expression were evaluatedby immunoblot assay.

Inhibition of protein expression of biomarker molecules adipocytes inpresence or absence of composition 1B was evaluated in immunoblot assay.Briefly, equal amount of cell lysates proteins were resolved in 7.5%SDS-PAGE; thereafter, the proteins were transferred to nitrocellulosemembrane. After blocking the non-specific sites, the membrane wasincubated with either anti-PPARγ, or anti-CD36, or anti-aP2, oranti-β3AR, or anti-ADRP, or anti-perilipin antibody. Finally, thespecific immuno-reactive bands were developed with West-picochemiluminescent substrate (Pierce Biotechnology, IL, USA), and theimmunoblot images were recorded in a Kodak Image Station (Kodak, USA).Band intensities were calculated densitometrically and normalized withexpression of actin in respective samples. The data is summarized inFIG. 6.

Example 24 Down-Regulation of Production of Atherosclerotic MarkerProteins by Composition 1B

Experimental protocol: Production inhibition of atherosclerotic markerproteins such as CD36, monocytes chemoattractant protein-1 (MCP-1), andoxidized Low density lipoprotein (Ox-LDL) by composition-1B wasevaluated in high glucose induced J774, mouse macrophage cells. Briefly,the cells were cultured in DMEM with 2 mM Glutamine, 100 U/mLpenicillin, 100 mg/mL streptomycin and 10% fetal bovine serum (Hyclone,Logan, Utah). Equal number of cells was seeded into 35 mm petri dishes(Corning, USA) one day before the experiment. The culture media wasreplaced with fresh, glucose free DMEM supplemented with 10% fetalbovine serum. Cells were pre-incubated with 5 ug/ml of eitherLI/DD-II/054A/01 or AR 933 or composition 1B for 2 h at 37° C. with 5%CO2, and then incubated with 600 mg/dL of glucose for 5 days. Thecontrol culture was supplemented with 100 mg/dL glucose. The cells wereharvested and lysed with lysis buffer. Cell lysates were clarified at14,000 g. Protein concentration was measured by Bradford method.

Inhibition of marker proteins expression in high glucose induced J774macrophage cells in presence or absence of composition 1B was evaluatedin immunoblot assay. Briefly, equal amount of cell lysates proteins wereresolved in 7.5% SDS-PAGE; thereafter, the proteins were transferred tonitrocellulose membrane. After blocking the non-specific sites, themembrane was incubated with either CD36, or MCP-1 or Ox-LDL specificantibody (R&D Systems, Minneapolis, Minn.). Finally, the specificimmuno-reactive bands were developed with West-pico chemiluminescentsubstrate (Pierce Biotechnology, IL, USA), and the immunoblot imageswere recorded in a Kodak Image Station (Kodak, USA). Band intensitieswere calculated densitometrically and normalized with expression ofactin in respective samples. The results are summarized in FIG. 7.

Example 25 Modulation of Adiponectin by LI/DD-II/054A/01, AR 933 andComposition 1B

Modulation of adiponectin protein by LI/DD-II/054A/01 or AR 933 orcomposition-1B in 3T3-L1 adipocytes was evaluated in Western immunoblotassay. The cell culture, treatment protocol and immunoblot assaymethodology were the same as described in Example 23. FIG. 8 summarizesthe enhancement of adiponectin protein expression in 3T3-L1 matureadipocytes by composition 1B or its individual components such asLI/DD-II/054A/01 or AR 933.

Example 26

Down Regulation of Protein Tyrosine Phosphatase-1B (PTP-1B) in 3T3-L1Preadipocytes by LI/DD-II/054A/01 or AR 933 or Composition 1B:

The 3T3-L1 preadipocytes were cultured in Dulbecco's Modified EaglesMedium (DMEM) supplemented with 2 mM glutamine, 4.5 g/L glucose and 10%fetal bovine serum. Equal number of cells was plated in each 35 cm² cellculture dish overnight. Plates were washed with serum free and phenolred free DMEM, then the cultures were pre treated with 5 μg/ml of eitherLI/DD-II/054A/01 or AR 933 or composition 1B in FBS free and phenol redfree DMEM containing 0.2% BSA and 1 g/liter glucose. The cell lysatesproteins were extracted in cell lysis buffer and protein concentrationswere measured by Bradford reagent. The modulation of PTP-1B expressionin cell lysates were analyzed by immunoblot assay using anti-PTP-1Bantibody following the method as described in example 23. FIG. 9 showsdown regulation of PTP 1B protein expression in LI/DD-II/054A/01 or AR933 or composition 1B treated 3T3-L1 preadipocytes.

Example 27 In Vivo Efficacy of Sphaeranthus indicus Ethyl AcetateExtract (LI/DD-II/054A/01) Against Metabolic Disorders

Efficacy of the Sphaeranthus indicus ethyl acetate extract(LI/DD-II/054A/01) was tested in high fat, high cholesterol, high saltand high sucrose diet induced model of metabolic syndrome.

Induction: A batch of 12 Sprague Dawley Rats was randomly divided into 2groups, each comprised of 6 animals. Animals were acclimatized for 7days prior to study initiation. Metabolic syndrome was induced byfeeding the rats with the metabolic syndrome diet containing 32 g ofroasted bengal gram, 27 g of sucrose, 17 g of milk powder, 5 g ofmineral salt mixture, 1 g of yeast, 2 g of butter, 11 g of groundnut oiland 5 g of cholesterol per 100 g of the diet for 8 weeks.

Treatment: Following 8 weeks induction phase, the animals were treatedorally (using oral feeding gavage) with allocated test substance orvehicle daily for 8 weeks. The treatment group animals were supplementedorally with 250 mg/kg body weight of LI/DD-II/054A/01 in 10 mL of 0.5%CMC in water for further 8 weeks. The control group of animals receivedonly the vehicle (10 mL of 0.5% CMC in water) during this period. Duringthe treatment phase, all animals were provided with the standard rodentdiet till the end of the study.

Body weights: Body weight of individual animal was recorded weekly forthe entire duration of the study. Mean body weights for the treatmentgroup and control group were determined. The body weight gain wascalculated at the end of 1^(st) week, 4^(th) week and 8^(th) week afterinitiation of treatment in comparison to initial body weight. Incomparison to the control group, LI/DD-II/054A/01 at 250 mg/kg doseexhibited highly potent and statistically significant (p<0.01) reductionin body weight gain (66.04%) in comparison to control group. The resultsof body weight gain for the treatment groups and control group aresummarized in FIGS. 10A and 10B.

Fat tissue weight: Abdominal and epididymal fat were isolated andweighed at the termination of the study and the results were representedin Table-9. Abdominal and epididymal fat weights in the treatment groupare lower, when compared to those in the control group. The total fatwas significantly reduced (p<0.05) in the treatment group supplementedwith LI/DD-II/054A/01.

Weight of Fat Tissues Isolated from Abdomen and Epididymal Area of Rats.

TABLE 9 Treatment Abdominal fat (g) Epididymal fat (g) Total fat (g)Control 4.52 ± 1.16 4.18 ± 1.56 8.70 ± 2.52 (10 mL/kg) LI/DD- 2.28 ±0.78 3.07 ± 0.74 5.36 ± 0.89 II/054A/01 (250 mg/kg) Values expressed asmean weight ± SD

Values expressed as mean weight±SD

Serum Biochemistry: Blood sampling was done via sinus orbital plexusunder mild anesthesia, before induction, before initiation of treatmentand after completion of treatment. Various biochemical parametersincluding lipid profile were evaluated using biochemistry reagentssupplied by Human, Germany, in an automated clinical chemistry analyzerHumaStar300, Make: Human, Germany. Mean values of the biochemicalparameters especially serum cholesterol levels and triglycerides levelswere estimated before induction, after induction/before treatment andafter treatment. Supplementation of LI/DD-II/054A/01 at 250 mg/kgresulted in improvement in fat profile with 15.3, 12.7 and 22.9percentage reductions respectively in serum cholesterol, LDL andtriglycerides.

Estimation of Biomarker Adiponectin: The serum adiponectin concentrationfor the control and treatment groups of animals were assessed usingdouble antibody based sandwich rat adiponectin ELISA kit. The assay wasperformed following the instructions provided by the manufacturer (LincoResearch, USA). The sensitivity of the assay is 0.155 ng/ml. Adiponectinassay revealed that supplementation of LI/DD-II/054A/01 at a dose of 250mg/day/kg body weight for 8-weeks resulted in significant (p=0.00618)improvement in serum adiponectin concentration, in comparison with thebaseline. The control group, however, did not show improvement in serumadiponectin concentration. The results are summarized in FIG. 11.

The Homeostasis Model Assessment (HOMA): The HOMA index was calculatedbased on serum insulin and glucose levels, using the following formula:Fasting insulin concentration (μU/mL)×Fasting glucose concentration(mmol/L)/22.5.

The supplementation of treatment group of rats with a daily dose of 250mg/kg body weight for 8-week treatment period resulted in significantreduction of HOMA index compared to control group. The data is presentedin FIG. 12.

Example 28 Synergistic Anti-Obesity Activity of Composition 1DComprising Sphaeranthus indicus Methanol Extract (LI/DD-II/054A/03) andGarcinia mangostana Methanol Extract (AR 933) in 3:1 Ratio

Efficacy of LI/DD-II/054A/03, AR 933 and composition 1D were testedagainst High Fat Diet induced obesity model of Sprague-Dawley rats.

Induction: Selected healthy Sprague-Dawley rats were randomly assignedto control or various treatment groups (n=7). All the animals allocatedin the obesity study were made experimentally obese through dietaryintervention during the entire eight weeks induction period by feedinghigh fat diet ad libitum containing Bengal gram 32 g, Wheat floor 15 g,Yeast 1 g, Butter 2 g, Ground nut oil 8 g, Casein 5 g, Vanaspathi 20 g,Vitamin mix 05 g, Milk powder 12 g and Mineral Salt mixture 4.5 g per100 g of high fat diet.

Treatment: Following 8 weeks of induction phase, the animals weretreated orally (using oral feeding gavage) with allocated testsubstances or vehicle daily for 8 weeks. The animals of treatment groupswere supplemented with 100 mg or 250 mg/kg body weight ofLI/DD-II/054A/03 or 250 mg/kg body weight of AR 933 or 250 mg/kg bodyweight of composition 1D in 10 mL of 0.5% CMC in water for further 8weeks. The control group of animals received only the vehicle (10 mL of0.5% CMC in water) during this period. During the treatment phase, allanimals were provided with the standard rodent diet till the end of thestudy.

Body weights: Body weight of individual animal was recorded weeklyduring the entire duration of the study. Mean body weights for thetreatment group and control group were determined. The body weight gainwas calculated at the end of 1^(st) week, 4^(th) week and 8^(th) weekafter initiation of treatment in comparison to initial body weights.LI/DD-II/054A/03 dose dependently inhibited the body weight gain in highfat diet induced obese rats. It exhibited 46.3% reduction in body weightgain in the treatment group supplemented with 100 mg/kg body weight ofLI/DD-II/054A/03. AR 933 and LI/DD-II/054A/03 at a dose of 250 mg/kgexhibited 40% and 80.1% reductions in body weight gain respectively.However, the composition 1D at the same dose level i.e. at 250 mg/kgexhibited significantly better reduction in body weight gain (89%)compared to its individual ingredients. The results of body weight gainfor the treatment groups and control group are summarized in FIG. 13

Food and water consumption were recorded daily and fasting blood sampleswere collected before initiation, after 4^(th) week and 8^(th) week(termination) of the study.

Those of ordinary skill in the art will appreciate that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments or examplesdisclosed, but is intended to cover modifications within the objectivesand scope of the present invention as defined in the specification.

We claim:
 1. A biologically active composition for treatment of obesity,comprising: an herbal component consisting of: a.) and b.), wherein a.)is from 20 to 80 wt. % of a solvent extract of Sphaeranthus indicusflower heads, wherein said solvent extract is obtained by extractionwith a solvent selected from the group consisting of: hexane,dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol,n-butanol, isopropanol, methyl isobutyl ketone, and mixtures thereof;and wherein b.) is from 80 to 20 wt. % of an organic solvent or aqueousorganic solvent extract of Garcinia mangostana fruit rind; and,optionally, at least one third component selected from the groupconsisting of: biologically active components from plants, animals andmicroorganisms, pharmaceutically acceptable active ingredients,vitamins, minerals, vehicles, carriers, diluents, and mixtures thereof.2. A biologically active composition for treatment of obesity,comprising: an herbal component consisting of: a.) and b.), wherein a.)is from 20 to 80 wt. % of an organic solvent or aqueous organic solventextract of Sphaeranthus indicus flower heads, wherein said organicsolvent or aqueous organic solvent extract contains from 8% to 21%7-hydroxyfrullanolide; and wherein b.) is from 80 to 20 wt. % of anorganic solvent or aqueous organic solvent extract of Garciniamangostana fruit rind and, optionally, at least one third componentselected from the group consisting of: biologically active componentsderived from plants, animals and microorganisms, pharmaceuticallyacceptable active ingredients, vitamins, minerals, vehicles, carriers,diluents, and mixtures thereof.
 3. The biologically active compositionof claim 1, wherein said composition further comprises at least oneexcipient.
 4. The biologically active composition of claim 2, whereinsaid composition further comprises at least one excipient.
 5. Thebiologically active composition of claim 1, wherein said treatment ofobesity comprises at least one of: promoting lipolysis; inhibitingadipogenesis; inhibiting lipid accumulation; inhibiting a digestiveenzyme selected from the group consisting of alpha-amylase andalpha-glucosidase; and inhibiting at least one biomarker associated withadipocyte generation.
 6. The biologically active composition of claim 2,wherein said treatment of obesity comprises at least one of: promotinglipolysis; inhibiting adipogenesis; inhibiting lipid accumulation;inhibiting a digestive enzyme selected from the group consisting ofalpha-amylase and alpha-glucosidase; and inhibiting at least onebiomarker associated with adipocyte generation.
 7. A method of treatingobesity in a patient in need thereof, comprising administering thebiologically active composition of claim 1 to said patient.
 8. A methodof treating obesity in a patient in need thereof, comprisingadministering the biologically active composition of claim 2 to saidpatient.
 9. A biologically active composition for treatment of metabolicsyndrome, comprising: an herbal component consisting of: a.) and b.),wherein a.) is from 20 to 80 wt. % of a solvent extract of Sphaeranthusindicus flower heads, wherein said solvent extract is obtained byextraction with a solvent selected from the group consisting of: hexane,dichloromethane, chloroform, ethyl acetate, acetone, methanol, ethanol,n-butanol, isopropanol, methyl isobutyl ketone, and mixtures thereof;and wherein b.) is from 80 to 20 wt. % of an organic solvent or aqueousorganic solvent extract of Garcinia mangostana fruit rind and,optionally, at least one third component selected from the groupconsisting of: biologically active components from plants, animals andmicroorganisms, pharmaceutically acceptable active ingredients,vitamins, minerals, vehicles, carriers, diluents, and mixtures thereof.10. A biologically active composition for treatment of at least one ofmetabolic syndrome, diabetes, and atherosclerosis, comprising: an herbalcomponent consisting of: a.) and b.), wherein a.) is from 20 to 80 wt. %of an organic solvent or aqueous organic solvent extract of Sphaeranthusindicus flower heads, wherein said organic or aqueous organic extractcontains from 8% to 21% 7-hydroxyfrullanolide; and wherein b.) is from80 to 20 wt. % of one organic solvent or aqueous organic solvent extractof Garcinia mangostana fruit rind; and, optionally, at least one thirdcomponent selected from the group consisting of: biologically activecomponents from plants, animals and microorganisms, pharmaceuticallyacceptable active ingredients, vitamins, minerals, vehicles, carriers,diluents, and mixtures thereof.
 11. The biologically active compositionof claim 1, wherein said at least one extract of Sphaeranthus indicuscomprises at least one frullanolide or eudesmanoid sesquiterpenecompound.
 12. The biologically active composition of claim 1, whereinsaid at least one extract of Sphaeranthus indicus comprises at least onefrullanolide or eudesmanoid sesquiterpene compound selected from thegroup consisting of: 7-hydroxyfrullanolide;11α,13-dihydro-3α,7α-dihydroxy-4,5-epoxy-6β,7-eudesmanolide;11α,13-dihydro-7α-acetoxy-3β-hydroxy-6β,7-eudesm-4-enolide;3-keto-β-eudesmol;11α,13-dihydro-3α,7α-dihydroxyeudesm-4-en-6α,12-olide;11α,13-dihydro-3α,7α-dihydroxy frullanolide;11α,13-dihydro-7α,13-dihydroxy frullanolide;11α,13-dihydro-7α-hydroxy-13-methoxyfrullanolide;2α,7α-dihydroxy-4-en-11,13-dihydroeudesm-6,12-olide; 2α-hydroxycosticacid; 3-keto-7α-hydroxyeudesm-4-en-6,12-olide (cryptomeridiol);4-epicryptomeridiol; sphaeranthanolide; 2α-hydroxysphaerantholide;2α-acetoxysphaerantholide; 2α,7α-dihydroxysphaerantholide;2α-acetoxy-7α-hydroxysphaerantholide; and2α-acetoxy-5α-hydroxyisosphaerantholide, or mixtures thereof.
 13. Thebiologically active composition of claim 11, wherein said at least oneextract of Sphaeranthus indicus comprises said at least one frullanolideor eudesmanoid sesquiterpene compound in a concentration in the range of0.01 to 95%.
 14. The biologically active composition of claim 1, whereinsaid at least one organic solvent or aqueous organic solvent extract ofGarcinia mangostana comprises α-mangostin, γ-mangostin, or a mixturethereof.
 15. The biologically active composition of claim 1, whereinsaid at least one organic solvent or aqueous organic solvent extract ofGarcinia mangostana comprises: α-mangostin in an amount of from 0.0010%to 99.9%; γ-mangostin in an amount of from 0.0010% to 99.9%; or amixture of α-mangostin and γ-mangostin in an amount of from 0.001% to99.9%.
 16. The biologically active composition of claim 1, wherein saidat least one organic solvent or aqueous organic solvent extract ofGarcinia mangostana comprises: α-mangostin in an amount of from 0.01% to95%; γ-mangostin in an amount of from 0.01% to 95%; or a mixture ofα-mangostin and γ-mangostin in an amount of from 0.01% to 95%.
 17. Thebiologically active composition of claim 1, wherein said third componentis at least one biologically active component selected from: extracts,fractions, or compounds, phytochemicals, or powders derived from plants,animals or microorganisms; and wherein said at least one biologicallyactive component has a health benefit selected from the group consistingof: anti-diabetic activity, anti-hyperglycemic activity, hypolipidemicactivity, anti-obesity activity, anti-hypertensive activity,anti-platelet aggregation activity, anti-infective activity,anti-atherosclerotic activity; and antiinflammatory activity,anti-oxidant activity and bio-enhancing activity.
 18. The biologicallyactive composition of claim 1, wherein said at least one third componentis selected from the group consisting of: vehicles, carriers, diluents,and mixtures thereof.
 19. The biologically active composition of claim18, wherein said vehicles, carriers, diluents and mixtures thereof areselected from the group consisting of: solid carriers selected from thegroup consisting of: glucose, fructose, sucrose, maltose, yellowdextrin, white dextrin, aerosol, microcrystalline cellulose, calciumstearate, magnesium stearate, sorbitol, stevioside, corn syrup, lactose,citric acid, tartaric acid, malic acid, succinic acid, lactic acid,L-ascorbic acid, dl-alpha-tocopherol, glycerin, propylene glycol,glycerin fatty ester, poly glycerin fatty ester, sucrose fatty ester,sorbitan fatty ester, propylene glycol fatty ester, acacia, carrageenan,casein, gelatin, pectin, agar, vitamin B group, nicotinamide, calciumpantothenate, amino acids, calcium salts, pigments, flavors, andpreservatives; liquid carriers or diluents or excipients selected fromthe group consisting of: distilled water, saline, aqueous glucosesolution, ethanol, propylene glycol, and polyethylene glycol; and oilycarriers selected from the group consisting of: animal and vegetableoils, white soft paraffin, paraffin, and wax.
 20. The biologicallyactive composition of claim 1, wherein said composition is provided asan oral dosage form, a topical formulation, a transdermal formulation, aparenteral dosage form, or an inhalable dosage form, suitable foradministration to a mammal in need thereof.
 21. The biologically activecomposition of claim 1, wherein said composition is formulated as: anoral dosage form selected from the group consisting of: tablets, softcapsules, hard capsules, pills, granules, powders, emulsions,suspensions, syrups, pellets, food, and beverages; a parenteral, dosageform selected from the group consisting of an injection solution, drops,or suppositories; a transdermal dosage form selected from the groupconsisting of patches, topical creams, and gel; or food ingredients orbeverages.